UNIVERSITY - MADHYA PRADESH BHOJ OPEN UNIVERSITY

BHOPAL (M.P.)

PROGRAMME - M.Sc.Chemistry (Previous)

PAPER - v (A-II)

TITLE OF PAPER - BIOLOGY FOR CHEMIST

BKOCK NO . - I

UNIT WRITER - UNIT - I Smt. Shikha Mandloi

Asst. Prof. Microbiology

Sri Sathya Sai College for Women

EDITOR - Dr.(Smt.) Renu Mishra, HOD, Botany & Microbiology,

Sri Sathya Sai College for Women, Bhopal

COORDINATION

COMMITTEE - Dr. Abha Swarup, Director, Printing & Translation

Major PradeepKhare, Consultant, Printing & Translation

1

POST GRADUATE PROGRAMME

M.Sc.CHEMISTRY (PREVIOUS)

DISTANCE EDUCATION SELF INSTRUCTIONAL MATERIAL

Paper-V(A-II)

BIOLOGY FOR CHEMISTS

BLOCK :I

UNIT—I : CELL STRUCTURE AND FUNCTION

MADHYA PRADESH BHOJ OPEN UNIVERSITY

BHOPAL (M.P.)

2

Cell structure and function

Introduction

The cell is the basic unit of life. There are millions of different types of cells. There are

cells that are organisms onto themselves, such as microscopic amoeba and bacteria cells. And

there are cells that only function when part of a larger organism, such as the cells that make up

your body. The cell is the smallest unit of life in our bodies. In the body, there are brain cells, skin

cells, liver cells, stomach cells, and the list goes on. All of these cells have unique functions and

features. And all have some recognizable similarities. All cells have an outer covering called the

plasma membrane, protecting it from the outside environment. The cell membrane regulates the

movement of water, nutrients and wastes into and out of the cell. Inside of the cell membrane are

the working parts of the cell. At the center of the cell is the cell nucleus. The cell nucleus contains

the cell's DNA, the genetic code that coordinates protein synthesis. In addition to the nucleus,

there are many organelles inside of the cell - small structures that help carry out the day-to-day

operations of the cell. One important cellular organelle is the ribosome. Ribosomes participate in

protein synthesis. The transcription phase of protein synthesis takes places in the cell nucleus.

After this step is complete, the mRNA leaves the nucleus and travels to the cell's ribosomes, where

translation occurs. Another important cellular organelle is the mitochondrion. Mitochondria

(many mitochondrion) are often referred to as the power plants of the cell because many of the

reactions that produce energy take place in mitochondria. Also important in the life of a cell are

the lysosomes. Lysosomes are organelles that contain enzymes that aid in the digestion of nutrient

molecules and other materials

There are many different types of cells. One major difference in cells occurs between plant

cells and animal cells. While both plant and animal cells contain the structures discussed above,

plant cells have some additional specialized structures. Many animals have skeletons to give their

body structure and support. Plants have a unique cellular structure called the cell wall. The cell

wall is a rigid structure outside of the cell membrane composed mainly of the polysaccharide

cellulose. The cell wall gives the plant cell a defined shape which helps support individual parts of

plants. In addition to the cell wall, plant cells contain an organelle called the chloroplast. The

chloroplast allow plants to harvest energy from sunlight. Specialized pigments in the chloroplast

(including the common green pigment chlorophyll) absorb sunlight and use this energy to complete

the chemical reaction.

3

UNIT-I

Cell structure and function

1.0 Introduction

1.1 Objectives

1.2 Structure of prokaryotic and eukaryotic cells

1.3 Intracellular organelles and their functions

1.4 Comparision of plant and animal cells

1.5 Over view of metabolism: catabolism and anabolism

1.6 ATP- the biological energy currency

1.7 Origin of life;-unique properties of carbon, chemical evolution and rise of living

systems

1.8 Introduction to biomolecules

1.9 Building blocks of biomacromolecules

2.0 Let us sum up

2.1Check your progress The key

2.2 Assignment/ Activity

2.3 References

4

1.0 Introduction

The cell is the basic unit of life. There are millions of different types of cells.

There are cells that are organisms onto themselves, such as microscopic amoeba and

bacteria cells. And there are cells that only function when part of a larger organism,

such as the cells that make up your body. The cell is the smallest unit of life in our

bodies. In the body, there are brain cells, skin cells, liver cells, stomach cells, and

the list goes on. All of these cells have unique functions and features. And all have

some recognizable similarities. All cells have an outer covering called the plasma

membrane, protecting it from the outside environment. The cell membrane

regulates the movement of water, nutrients and wastes into and out of the cell. Inside

of the cell membrane are the working parts of the cell. At the center of the cell is the

cell nucleus. The cell nucleus contains the cell's DNA, the genetic code that

coordinates protein synthesis. In addition to the nucleus, there are many organelles

inside of the cell - small structures that help carry out the day-to-day operations of

the cell. One important cellular organelle is the ribosome. Ribosomes participate in

protein synthesis. The transcription phase of protein synthesis takes places in the

cell nucleus. After this step is complete, the mRNA leaves the nucleus and travels to

the cell's ribosomes, where translation occurs. Another important cellular organelle

is the mitochondrion. Mitochondria (many mitochondrion) are often referred to as

the power plants of the cell because many of the reactions that produce energy take

place in mitochondria. Also important in the life of a cell are the lysosomes.

Lysosomes are organelles that contain enzymes that aid in the digestion of nutrient

molecules and other materials. Below is a labelled diagram of a cell to help you

identify some these structures.

5

There are many different types of cells. One major difference in cells occurs

between plant cells and animal cells. While both plant and animal cells contain the

structures discussed above, plant cells have some additional specialized structures.

Many animals have skeletons to give their body structure and support. Plants have a

unique cellular structure called the cell wall. The cell wall is a rigid structure outside

of the cell membrane composed mainly of the polysaccharide cellulose. The cell wall

gives the plant cell a defined shape which helps support individual parts of plants. In

addition to the cell wall, plant cells contain an organelle called the chloroplast. The

chloroplast allow plants to harvest energy from sunlight. Specialized pigments in the

6

chloroplast (including the common green pigment chlorophyll) absorb sunlight and

use this energy to complete the chemical reaction:

6 CO2 + 6 H2O + energy (from sunlight) C6H12O6 + 6 O2

In this way, plant cells manufacture glucose and other carbohydrates that they can

store for later use.

1.1 Objectives

1. This unit will fulfill the basic introduction , function, origin and chemical

composition of some organelles.

2. Students will find the text useful as it will help in understanding some

molecular diagnostic technique.

1.3 Structure of prokaryotic and eucaryotic cell

Organisms contain many different types of cells that perform many different

functions There are two types of cells: eukaryotic and prokaryotic. Prokaryotic

cells are usually independent, while eukaryotic cells are often found in

multicellular organisms.

Prokaryotic cells

The prokaryote cell is simpler, and therefore smaller, than a eukaryote cell, lacking a

nucleus and most of the other organelles of eukaryotes. There are two kinds of

prokaryotes: bacteria and archaea; these share a similar structure.

7

Nuclear material of prokaryotic cell consist of a single chromosome which is in

direct contact with cytoplasm. Here the undefined nuclear region in the cytoplasm is

called nucleoid.

A prokaryotic cell has three architectural regions:

On the outside, flagella and pili project from the cell's surface. These are

structures (not present in all prokaryotes) made of proteins that facilitate

movement and communication between cells;

Enclosing the cell is the cell envelope – generally consisting of a cell wall

covering a plasma membrane though some bacteria also have a further

covering layer called a capsule. The envelope gives rigidity to the cell and

separates the interior of the cell from its environment, serving as a protective

filter. Though most prokaryotes have a cell wall, there are exceptions such as

Mycoplasma (bacteria) and Thermoplasma (archaea). The cell wall consists of

peptidoglycan in bacteria, and acts as an additional barrier against exterior

forces. It also prevents the cell from expanding and finally bursting (cytolysis)

from osmotic pressure against a hypotonic environment. Some eukaryote cells

(plant cells and fungi cells) also have a cell wall;

Inside the cell is the cytoplasmic region that contains the cell genome (DNA)

and ribosomes and various sorts of inclusions. A prokaryotic chromosome is

usually a circular molecule (an exception is that of the bacterium Borrelia

burgdorferi, which causes Lyme disease). Though not forming a nucleus, the

DNA is condensed in a nucleoid. Prokaryotes can carry extrachromosomal

DNA elements called plasmids, which are usually circular. Plasmids enable

additional functions, such as antibiotic resistance.

8

Structures outside the cell wall

Capsule

A gelatinous capsule is present in some bacteria outside the cell wall. The capsule

may be polysaccharide as in pneumococci, meningococci or polypeptide as Bacillus

anthracis or hyaluronic acid as in streptococci Capsules are not marked by ordinary

stain and can be detected by special stain. The capsule is antigenic. The capsule has

antiphagocytic function so it determines the virulence of many bacteria. It also plays

a role in attachment of the organism to mucous membranes.

Flagella

Flagella are the organelles of cellular mobility. They arise from cytoplasm and

extrude through the cell wall. They are long and thick thread-like appendages,

protein in nature. Are most commonly found in bacteria cells but are found in animal

cells as well.

Fimbriae (pili)

They are short and thin hair like filaments, formed of protein called pilin (antigenic).

Fimbriae are responsible for attachment of bacteria to specific receptors of human

cell (adherence). There are special types of pili called (sex pili) involved in

conjunction.

9

BACTERIAL CELL:-AN EAXMPLE OF PROCARYOTIC CELL

Eukaryotic cells

Eukaryotic cells are about 15 times wider than a typical prokaryote and can be as

much as 1000 times greater in volume. The major difference between prokaryotes

and eukaryotes is that eukaryotic cells contain membrane-bound compartments in

which specific metabolic activities take place. Most important among these is a cell

nucleus, a membrane-delineated compartment that houses the eukaryotic cell's DNA.

10

This nucleus gives the eukaryote its name, which means "true nucleus." Other

differences include:

The plasma membrane resembles that of prokaryotes in function, with minor

differences in the setup. Cell walls may or may not be present.

The eukaryotic DNA is organized in one or more linear molecules, called

chromosomes, which are associated with histone proteins. All chromosomal

DNA is stored in the cell nucleus, separated from the cytoplasm by a

membrane. Some eukaryotic organelles such as mitochondria also contain

some DNA.

Many eukaryotic cells are ciliated with primary cilia. Primary cilia play

important roles in chemosensation, mechanosensation, and thermosensation.

Cilia may thus be "viewed as sensory cellular antennae that coordinate a large

number of cellular signaling pathways, sometimes coupling the signaling to

ciliary motility or alternatively to cell division and differentiation."[7]

Eukaryotes can move using motile cilia or flagella. The flagella are more

complex than those of prokaryotes.

Table 1: Comparison of features of prokaryotic and eukaryotic cells

Prokaryotes Eukaryotes

Typical

organisms bacteria, archaea protists, fungi, plants, animals

Typical size ~ 1–10 µm ~ 10–100 µm (sperm cells, apart from the

tail, are smaller)

Type of nucleus nucleoid region; no

real nucleus real nucleus with double membrane

DNA circular (usually) linear molecules (chromosomes) with histone

11

proteins

RNA-/protein-

synthesis

coupled in

cytoplasm

RNA-synthesis inside the nucleus

protein synthesis in cytoplasm

Ribosomes 50S+30S 60S+40S

Cytoplasmatic

structure very few structures

highly structured by endomembranes and a

cytoskeleton

Cell movement flagella made of

flagellin

flagella and cilia containing microtubules;

lamellipodia and filopodia containing actin

Mitochondria none one to several thousand (though some lack

mitochondria)

Chloroplasts none in algae and plants

Organization usually single cells single cells, colonies, higher multicellular

organisms with specialized cells

Cell division Binary fission

(simple division)

Mitosis (fission or budding)

Meiosis

The cells of eukaryotes and prokaryotes .

All cells, whether prokaryotic or eukaryotic, have a membrane that envelops the cell,

separates its interior from its environment, regulates what moves in and out

(selectively permeable), and maintains the electric potential of the cell. Inside the

membrane, a salty cytoplasm takes up most of the cell volume. All cells possess

DNA, the hereditary material of genes, and RNA, containing the information

necessary to build various proteins such as enzymes, the cell's primary machinery.

There are also other kinds of biomolecules in cells. This article will list these primary

components of the cell, then briefly describe their function.

12

Cell membrane

The cytoplasm of a cell is surrounded by a cell membrane or plasma membrane. The

plasma membrane in plants and prokaryotes is usually covered by a cell wall. This

membrane serves to separate and protect a cell from its surrounding environment and

is made mostly from a double layer of lipids (hydrophobic fat-like molecules) and

hydrophilic phosphorus molecules. Hence, the layer is called a phospholipid bilayer.

It may also be called a fluid mosaic membrane. Embedded within this membrane is a

variety of protein molecules that act as channels and pumps that move different

molecules into and out of the cell. The membrane is said to be 'semi-permeable', in

that it can either let a substance (molecule or ion) pass through freely, pass through

to a limited extent or not pass through at all. Cell surface membranes also contain

receptor proteins that allow cells to detect external signaling molecules such as

hormones..

The cytoskeleton acts to organize and maintain the cell's shape; anchors organelles in

place; helps during endocytosis, the uptake of external materials by a cell, and

cytokinesis, the separation of daughter cells after cell division; and moves parts of

the cell in processes of growth and mobility. The eukaryotic cytoskeleton is

composed of microfilaments, intermediate filaments and microtubules. There is a

great number of proteins associated with them, each controlling a cell's structure by

directing, bundling, and aligning filaments. The prokaryotic cytoskeleton is less

well-studied but is involved in the maintenance of cell shape, polarity and

cytokinesis.

Genetic material

13

Two different kinds of genetic material exist: deoxyribonucleic acid (DNA) and

ribonucleic acid (RNA). Most organisms use DNA for their long-term information

storage, but some viruses (e.g., retroviruses) have RNA as their genetic material. The

biological information contained in an organism is encoded in its DNA or RNA

sequence. RNA is also used for information transport (e.g., mRNA) and enzymatic

functions (e.g., ribosomal RNA) in organisms that use DNA for the genetic code

itself. Transfer RNA (tRNA) molecules are used to add amino acids during protein

translation.

Prokaryotic genetic material is organized in a simple circular DNA molecule (the

bacterial chromosome) in the nucleoid region of the cytoplasm. Eukaryotic genetic

material is divided into different, linear molecules called chromosomes inside a

discrete nucleus, usually with additional genetic material in some organelles like

mitochondria and chloroplasts. A human cell has genetic material contained in the

cell nucleus (the nuclear genome) and in the mitochondria (the mitochondrial

genome). In humans the nuclear genome is divided into 23 pairs of linear DNA

molecules called chromosomes. The mitochondrial genome is a circular DNA

molecule distinct from the nuclear DNA. Although the mitochondrial DNA is very

small compared to nuclear chromosomes, it codes for 13 proteins involved in

mitochondrial energy production and specific tRNAs.

Foreign genetic material (most commonly DNA) can also be artificially introduced

into the cell by a process called transfection. This can be transient, if the DNA is not

inserted into the cell's genome, or stable, if it is. Certain viruses also insert their

genetic material into the genome.

.

14

1.3 INTRACELLULAR ORGANELLES AND THEIR FUNCTIONS

The plasma membrane is followed by the cytoplasm which is distinguished in to

A. cytosol and B cytoplasmic structures.

A The cytosol is the gelatinous fluid that fills the cell and surrounds the

organelles.

B cytoplasmic structures:- In the cytoplasmic matrix certain living and non living

structures remain suspended.The non living structures are called inclusions while

living structures are called organelles.

The human body contains many different organs, such as the heart, lung, and kidney,

with each organ performing a different function. Cells also have a set of "little

organs," called organelles, that are specialized for carrying out one or more vital

functions. There are several types of organelles in a cell. Some (such as the nucleus

and golgi apparatus) are typically solitary, while others (such as mitochondria,

peroxisomes and lysosomes) can be numerous (hundreds to thousands).

Golgi Apparatus

An Italian neurologist (i.e., physician) Camillo Golgi in 1873 discovered, them

which is commonly known as the Golgi bodies. For the performance of certain

important cellular functions such as biosynthesis of polysaccharides, packaging

(compartmentalizing) of cellular synthetic products (proteins), production of

exocytotic (secretory) vesicles and differentiation of cellular membranes, there

occurs a complex organelle called Golgi complex or Golgi apparatus in the in the

cytoplasm of animal and plant cells. The Golgi apparatus, like the endoplasmic

reticulum, is a canalicular system with sacs, but unlike the endoplasmic reticulum it

15

has parallely arranged, flattened, membrane-bounded vesicles which lack ribosomes

and stainable by osmium tetraoxide and silver salts.

Occurence

The Golgi apparatus occurs in all cells except the prokaryotic cells (viz.,

mycoplasmas, bacteria and blue green algae) and eukaryotic cells of certain fungi,

sperm cells of bryophytes and pteridiophytes, cells of mature sieve tubes of plants

and mature sperm and red blood cells of animals. Their number per plant cell can

vary from several hundred as in tissues of corn root and algal rhizoids (i.e., more

than 25,000 in algal rhizoids, Sievers, 1965), to a single organelle in some algae.

(Certain algal cells such as Pinularia and Microsterias, contain largest and most

complicated Golgi apparatuses. In higher plants, Golgi apparatuses are particularly

common in secretory cells and in young rapidly growing cells.

In animal cells, there usually occurs a single Golgi apparatus, however, its

number may vary from animal to animal and from cell to cell. Thus Paramoeba

species has two Golgi apparatus and nerve cells, liver cells and chordate oocytes

have multiple Golgi apparatuses, there being about 50 of them in the liver cells.

Distribution

In the cells of higher plants, the Golgi bodies or dictyosomes are usually found

scattered throughout the cytoplasm and their distribution does not seem to be ordered

or localized in any particular manner (Hall et al., 1974). However, in animal cells the

Golgi apparatus is a localized organelle. For example, in the cells of ectodermal or

endodermal origin, the Golgi apparatus remains polar and occurs in between the

nucleus and the periphery (e.g., thyroid cells, exocrine pancreatic cells and mucus-

producing goblet cells of intestinal epithelium) and in the nerve cells it occupies a

circum-nuclear position.

Structure

16

The Golgi apparatus is morphologically very similar in both plant and animal

cells. However, it is extremely pleomorphic: in some cell types it appears compact

and limited, in others spread out and reticular (net-like). Its shape and form may very

depending on cell type. Typically, however, Golgi apparatus appears as a complex

array of interconnecting tubules, vesicles and cisternae.The simplest unit of the Golgi

apparatus is the cisterna. This is a membrane bound space in which various materials

and secretions may accumulate. Numerous cisternae are associated with each other

and appear in a stack like (lamellar) aggregation. A group of these cisternae is called

the dictyosome, and a group of dictyosomes makes up the cells Golgi apparatus. All

dictyosomes of a cell have a common function.

1. Flattened Sac or Cisternae

Cisternae (aboute 1m in diameter) are central, flattened, plate-like or saucer-

like closed compartments which are help in parallel bundles or stacks on

above the other. In each stack, cisternae, are separated by a space of 20 to 30

nm which may contain rod-like elements or fibres. Each stack of cisternae

forms a dictyosome which may contain 5 to 6 Golgi cisternae in animal cells

or 20 or more cisternae in plant cells. Each cisterna is bounded by a smooth

unit membrane (7.5 nm thick), having a lumen varying in width from about

500 to 1000 nm The margins of each cisterna are gently curved so that the

entire dictyosome of Golgi apparatus takes on a bow like appearance. The

cisternae at the convex end of the dictyosome comprise proximal forming or

cis-face and the cisternae at the concave end of the dictyosome comprise the

distal, maturing or trans-face. The forming or cis-face of Golgi is located next

to either the nucleus or a specialized portion of rough ER that lacks bound

ribosomes and is called ''transitional'' ER. Trans face of Golgi is located near

17

the plasma membrane. This polarization is called cis-trans axis of the Golgi

apparatus.

2. Tubules

A complex array of associated vesicles and anastomosing tubules (30 to 50nm

diameter) surround the dictyosome and radiate from it. In fact, the peripheral

area of dictyosome is fenestrated (lace-like) in structure. 3.Vesicles

The vesicles (60 nm in diameter) are of three types :

(i) Transitional vesicles are small membrane limited vesicles which are

through to form as blebs from the transitional ER to migrate and

converge to cis face of Golgi, where they coalesce to form new

cisternae.

(ii) Secretory vesicles are varied-sized membrane-limited vesicles which

discharge from margins of cisternae of Golgi. They, often, occur

between the maturing face of Golgi and the plasma membrane.

(iii) Clathrin-coated vesicles are spherical protuberances, about 50m in

diameter and with a rough surface. They are found at the periphery of

the organelle, usually at the ends of single tubules, and are

morphologically quite distinct from the secretory vesicles. The clathrin-

coated vesicles are known to play a role in intra-cellular traffic or

membranes and of secretory products. i.e., between ER and Golgi, as

well as between GELR region and the endosomal and lysosomal

compartments.

Function

Golgi vesicles are often, referred to as the ''traffic police" of the cell (Dernell

et al., 1986. They play a key role in sorting many of cell's proteins and membrane

constituents, and in directing them to their proper destinations. To perform this

18

function, the Golgi vesicles contain different sets of enzymes in different types of

vesicles-cis, middle and trans cisternae- that react with and modify secretory proteins

passing through the Golgi lumen or membrane proteins and glycoprotein's in the

Golgi membranes as they are on route to their final destinations. For example a Golgi

enzyme may add a "signal" or "tag" such as a carbohydrate or phosphate residues to

certain proteins to direct them to their proper sites in the cell. Or, a proteolytic Golgi

enzyme may cut a secretory or membrane protein into two or more specific segments

(E.g., molecular processing involved in the formation of pancreatic hormone insulin:

preproinsulinproinsulininsulin).

Recently, in the function of Golgi apparatus, sub compartmentalization with a

division of labour has been proposed between the cis region (in which proteins of

RER are sorted and some of them are returned back possibly by coated vesicles), and

the trans region in which the most refined proteins are further separated for their

delivery to the various cell compartments (e.g., plasma membrane, secretory

granules and lysosomes).

Thus, Golgi apparatus is a centre of reception, finishing, packaging, and dispatch for

a variety of materials in animal and plant cells :

1. Golgi Functions in Plants

19

In plants, Golgi apparatus is mainly involved in the secretion of mainly

involved in the secretion of materials of primary and secondary cell walls

(e.g., formation and export of glycoprotein, lipids, pectins and monomers for

hemicellulose, cellulose, lignin, etc). During cytokinesis of mitosis or meiosis,

the vesicles originating from the periphery of Golgi apparatus, coalesce in the

phragmoplast area to form a semisolid layer, called cell plate. The unit

membrane of Golgi vesicles fus During early electron microscopic studies,

rounded dense bodies were observed in rat liver cells. These bodies were

initially described as "perinulclear dense bodies", C. de Duve, in 1955,

renamed these organelles as "lysosomes" to indicate that the internal digestive

enzymes only became apparent when the membrane of these organelles was

lysed (See Reid and Leech, 1980). However, the term lysosome means lytic

body having digestive enzymes capable of lysis (viz., dissolution of a cell or

tissue es during cell plate formation and becomes part of plasma membrane of

daughter cells .

Lysosomes

The lysosomes (Gr. lyso=digestive + soma = bodies) are tiny membrane-bound

vesicles involved in intracellular digestion. They contain a variety of hydrolytic

enzymes that remain active under acidic conditions. The lysosomal lumen is

maintained at an acidic pH (around 5) by an ATP-driven proton pump in the

membrane. Thus, these remarkable organelles are primarily meant for the digestion

of a variety of biological materials and secondarily cause aging and death of animal

cells and also a variety of human diseases such as cancer, gout Pompe's silicosis and

I-cell disease.

20

Occurence

The lysosomes occur in most animal and few plant cells. They are absent in

bacteria and mature mammalian ertythrocytes. Few lysosomes occur in muscle cells

or in cells of the pancreas. Leucocytes, especially granulocytes are a particularly rich

source of lysosomes. Their lysosomes are so large sized that they can be observed

under the light microscope. Lysosomes are also numerous in epithelial cells of

absorptive, secretory and excretory organs (e.g., intestine, liver, kidney, etc.) They

occur in abundance in the epithelial cells of lungs and uterus. Lastly phagocytic cells

and cells of reticuloendothelial system (e.g., bone marrow, spleen and liver) are also

rich in lysosomes.

Structure

The lysosomes are round vacuolar structure which remain filled with dense

material and are bounded by single unit membrane. Their shape and density vary

greatly. Lysosmes are 0.2 to 0.5m in size. Since, size and shape of lysosomes vary

from cell to cell and time to time (i.e. they are polymorphic), their identification

becomes difficult. However, on the basis of the following three criteria, a cellular

entity can be identified as a lysosome : (1) It should be bound by a limiting

membrane (2) It should contain two or more acid hydrolases ; and (3) It should

demonstrate the property of enzyme latency when treated in away that adversely

affects organelle's membrane structure. Lysosomes are very delicate and fragile

organelles. Lysosomal fraction have been isolated by sucrose-density centrifugation

(or Isopycnic centrifugation) after mild methods of homogenization.

Lysosomes tend to accumulate certain dyes (vital stains such as Neutral red,

Niagara, Evans blue) and drugs such as anti-malarial drug chloroquine. Such 'loaded'

21

lysosomes can be demonstreated by fluorescence microscopy. The location of the

lysosomes in the cell can also be pinpointed by various histochemical or

cytochemical methods. Certain lysosomal enzymes are good histochemical markers.

For example, acid phosphatase is the principal enzyme which is used as a marker for

the lysosomes by the use of Gomori staining technique (Gomori, 1952). Specific

stains are also used for other lysosomal enzymes such as B-glucuronidase, aryl

sulphatatase, N-acetyl-B-glucosaminidase and 5-bromo-4-chloroindolacetate

esterase.

Lysosomal Enzymes

According to a recent estimate, a lysosome may contain up to 40 types of

hydrolytic enzymes (see Alberts et al., 1989). they include proteases (e.g., cathespsin

for protein digestion), nucleases, glycosidases (for digestion of polysaccharides and

glycosides ), lipase, phospholipases, phosphatases and sulphatases (table 8-2). All

lysosomal enzymes are acid hydrolases, optimally active at the pH5 maintained

within lysosomes. The lysosomal enzymes latent and out of the cytoplasmic matrix

or cytosol (whose pH is about ~ 7.2), but the acid dependency of lysosmal enzymes

protects the contents of the cytosol (cytoplasmic matrix) against any damage even if

leakage of lysosomal enzymes should occur.

The so-called latency of the lysosomal enzymes is due to the presence of the

membrane which is resistant to the enzymes that it encloses. Most probably this is to

the face that most lysosomal hydrolases are membrane-bound, which may prevalent

the active centre of enzymes to gain access to susceptible groups in the membrane (

Reid and Leech, 1980).

22

Kind of Lysosomes

Lysosomes are extremely dynamic organelles, exhibiting polymorphism in

their morphology. Following four types of lysosomes have been recognized in

different types of cells or at different time in the same cell. Of these, only the first is

the primary lysosome, the other three have been grouped together as secondary

lysosomes.

1. Primary Lysosomes

These are also called storage granules, protolysosomes or virgin lysosomes.

Primary lysosomes are newly formed organelles bounded by a single

membrane and typically having a diameter of 100nm. They contain the

degradative enzymes which have not participated in any digestive process.

Each primary lysosome contains one type of enzyme or another and it is only

in the secondary lysosome that the full complement of acid hydrolases is

present.

2. Heterophagosomes

They are also called heterophagic vacuoles, heterolysosomes or

phagolysosomes. Heterophagosomes are formed by the fusion of primary

lysosomes with cytoplasmic vacuoles containing extracellular particles into

the cell by any of a variety of endocytic processes (e.g., pinocytosis,

phagocytosis or receptor mediated endocytosis. The digestion of engulfed

substances takes place by the enzymatic activities of the hydrolytic enzymes of

the secondary lysosomes. The digested material has low molecular weight and

readily passes through the membrane of the lysosomes to become the part of

the matrix.

23

3. Autophagosomes

They are also called autophagic vacuole, cytolysosomes or autolysosomes.

Primary lysosomes are able to digest intracellular structures including

mitochondria, ribosomes, peroxisomes and glycogen granules. Such

autodigestion (called autophagy) of cellular organelles is a normal event

during cell growth and repair and is especially prevalent in differentiating and

de-differentiating tissues (e.g., cells undergoing programmed death during

meta-morphosis or regeneration) and tissue under stress. Autophagy takes

several forms. In some cases the lysosome appears to flow around the cell

structure and fuse, enclosing it in a double membrane sac, the lysosomal

enzymes being initially confined between the membrane. The inner membrane

then breaks down and the enzymes are able to penetrate to the enclosed

organelle. In other cases, the organelle to be digested is first encased by

smooth ER, forming a vesicle that fuses with a primary lysosome. Lysosomes

also regularly engulf bits of cytosol (cytoplasmic matrix) which is degraded by

a process, called microautophagy.

As digestion proceeds, it becomes increasingly difficult to identify the nature

of the original secondary lysosome (i.e., heterophagosome or autophagosome)

and the more general term digestive vacuole is used to describe the organelle

at this stage.

4. Residual Bodies

They are also called telolysosomes or dense bodies. Residual bodies are forced

if the digestion inside the food vacuole is incomplete. Incomplete digestion

may be due to absence of some lysosomal enzymes. The undigested food is

24

present in the digestive vacuole as the residues and may take the form of

whorls of membranes. grains, amorphouse masses, ferritin-like or myelin

figures.

Residual bodies are large, irregular in shape and are usually quite electron-

dense. In some cells, such as Amoeba and other potozoa, these residual bodies

are eliminated defecation. In other cells, residual bodies may remain for a long

time and may load the cells to result in their aging. For example, pigment

inclusions (age pigment or lipofuscin granules) found in nerve cells (also in

liver cells, heart cells and muscle cells) of old animals may be due to the

accumulation of residual bodies.

Origin

The biogenesis (origin) of the lysosomes requires the synthesis of specialized

lysosomal hydrolases and membrane proteins. Both classes of proteins are

synthesized in the ER and transported through the Golgi apparatus, then transported

from the trans Golgi network to an intermediate compartment (an endolysosome) by

means of transport vesicles (which are coated by clathrin protein). The lysosmal

enzymes are glycol proteins, containing N-linked oligosaccharides that are processed

in a unique way in the cis Golgi so that their mannose residues are phophorylated.

These transport vesicles containing the M6P-receptors act as shuttles that move the

receptors back and forth between the Golgi network and endolysosomes. They low

pH in the endolysosome dissociates the lysosomal hydrolases from this receptor,

making the transport of the hydrolases unidirectional.

Function of Lysosomes

The important functions of lysosomes are as follow :

25

1. Digestion of large extracellular particles. The lysosomes digests the food

contents of the phoagosomes or pinosomes.

2. Digestion of intracellular substance. During the starvation, the lysosomes

digest the stored food contents viz proteins, lipids and carbohydrates

(glycogen) of the cytoplasm and supply to the cell necessary amount of

energy.

3. Autolysis. In certain pathological conditions the lysosomes start to digest the

various organelles of the cells and this process is known as autolysis or

cellular autophagy. When a cell dies, the lysosome membrane ruptures and

enzymes are liberated. These enzymes digest the dead cells. In the process of

metamorphosis of amphibians and / tunicates many embryonic tissues, e.g.,

gills, fins, tail, etc., are digested by the lysosomes and utilized by the other

cells.

4. Extracellular Digestion. The lysosomes of certain cells such as sperms

discharge their enzymes outside the cell during the process of fertilization. The

lysosomal enzymes digest the limiting membrane of the ovum and form

penetration path in ovum for the sperms. Acid hydrolases are released from

osteoclasts and break down bone for the reabsorption ; these cells also secrete

lactic acid which makes the local pH enough for optimal enzyme activity.

Likewise, preceding ossification (bone formation), fibroblasts release

cathepsin D enzyme to break down the connective tissue.

26

Lysosomes in plants

Plants contain several hydrolases, but they are not always as neatly

compartmentalized as they are in animal cells. Many of these hydrolases are found

bound to and functioning within the vicinity of the cells wall and are not necessarily

contained in membrane bound vacuoles at these sites. Many types of vacuoles and

storage granules of plants are found to contain certain digestive enzymes and these

granules are considered as lysosomes of plant cell (Gahan, 1972). According to

Matile (1969) the plant lysosomes can be defined as membrane bound cell

compartments containing hydrolytic digestive enzymes. Matile (1975) has divided

vacuoles of plants into following three types :

1. Vacuoles

The vacuole of a mature plant cell is formed from the enlargement and fusion

of smaller vacuoles present in meristematic cells; these provacuolse, which are

27

believed to be derived from the ER and possibly the Golgi and contain acid

hydrolases. These lysosomal enzymes are associated with the tonoplast of

large vacuole of differentiating cells. Sometimes, mitochondria and plastids

are observed inside the vacuole suggesting autophagy in plants (Swanson and

Webster,1989).

2. Spherosomes

The spherosomes are membrane bounded, spherical particles of 0.5 to 2.5 m

diameter, occurring in most plant cells. They have a fine granular structure

internally which is rich in lipids and proteins. They originate from the

endoplasmic reticulum (ER). Oil accumulates at the end of a strand of ER and

a small vesicle is then cut off by contribution to form particles, called

prospherosomes. The prospherosomes grow in size to form spherosoms.

Basiocally, the spherosomes are involved in lipid synthesis and storage. But,

the spherosomes of maize root tips (Matile, 1968) and spherosome of tobacco

endosperm tissue (Spichiger, 1969) have been found rich in hydrolytic

digestive enzymes and so have been considered as lysosomes. Like lysosomes

they are not only responsible for the accumulation and mobilization of reserve

lipids, but also for the digestion of other cytoplasmic components incorporated

by phagocytosis.

3. Aleurone Grain

The aleurone grains or protein bodies are spherical membrane-bounded

storage particle occurring in the cells of endosperm and cytoledons of seeds.

They are formed during the later stages of seed ripening and disappear in the

early stages of germination. They store protein (e.g., globulins) and phosphate

28

in the form of phytin. Matile (1968) has demonstrated that aleurone grains

from pea seed contain a wide range of hydrolytic enzymes including protease

and phosphatase which are required for the mobilization of stored protein and

phosphate, although the presence of other enzymes such as -amylase and

RNAase suggest that other cell constituents may also be digested. Thus like

spherosomes, aleurone grains store reserve materials, mobilize them during

germination and in addition form a compartment for the digestion of other cell

components (Hall et al., 1974). The aleurone grains are derived from the

strands of the endoplasmic reticulum.

During germinating of barley seed, the activity of hydrolases is found to be

controlled by hormones such as gibberellic acid. Gibberellic acid, a plant

growth hormone, is released by the embryo to the aleurone layer where, in

turn, the hyrolases are released to the endosperm. This hormone operates by

derepressing appropriate genes in the aleurone cells, which then begin to crank

out new hydrolytic proteins .

Endoplasmic Reticulum

The cytoplasmic matrix is traversed by a complex network of inter-connecting

membrane bound vacuoles or cavities. often remain concentrated in the

endoplasmic portion of the cytoplasm ; therefore, known as endoplasmic reticulum,

a name derived from the fact that in the light microscope it looks like a "net in the

cytoplasm." (Eighteenth-century European ladies carried purses of netting called

reticules).

The name "endoplasmic reticulum" was coined in 1953 by Porter, who in 1945

had observed it in electron micrographs of liver cells. Fawcet and Ito (1958),

29

Thiery (1958) and Rose and Pomerat (1960) have made various important

contributions to the endoplasmic reticulum.

The occurrence of the endoplasmic reticulum varies from cell to cell. The

erythrocytes (RBC), egg and embryonic cells lack in endoplasmic reticulum.

The spermatocytes have poorly developed endoplasmic reticulum. The adipose

tissues, brown fat cells and adrenocortical cells, interstitial cells of testes and cells

of corpus luteum of ovaries, sebaceous cells and retinal pigment cells contain only

smooth endoplasmic reticulum (SER). The cells of those organs which are actively

engaged in the synthesis of proteins such as chinar cells of pancreas, plasma cells,

goblet cells and cells of some endocrine glands are found to contain rough

endoplasmic reticulum (RER) which is highly developed. The presence of both

SER and RER in the hepatocytes (liver cells) is reflective of the variety of the roles

played by the liver in metabolism.

Morphology

Morphologically, the endoplasmic reticulum may occur in the following three

forms : 1. Lamellar form or cisternae (A closed, fluid-filled sac, vesicle or cavity is

called cisternae) 2. vesicular form or vesicle and 3. tubular form or tubules.

1. Cisternae. The cisternae are long, flattened, sac-like, unbranched tubules

having the diameter of 40 to 50 m. They remain arranged parallely in

bundles or stakes. PER usually exists as cisternae which occur in those

cells of pancreas, notochord and brain.

2. Vesicles. The vesicles are oval, membrane bound vacuolar structures

having the diameter of 25 to 500m. They often remain isolated in the

cytoplasm and occur in most cells but especially abundant in the SER.

30

3. Tubules. The tubules are branched structures forming the reticular system

along with the cisternae and vesicles. They usually have the diameter from

50 to 190m and occur almost in all the cells. Tubular form of ER is often

found in SER and is dynamic in nature, i.e., it is associated with membrane

movements, fission and fusion between membranes of cytocavity network

(see Thorpe, 1984).

Ultra structure

The cavities of cisternae, vesicles and tubules of the endoplasmic reticulum

are bounded by a thin membrane of 50 to 60 A0 thickness. The membrane of

endoplasmic reticulum is fluid-mosaic like the unit membrane of the plasma

membrane, nucleus, Golgi apparatus, etc. The membrane, thus, is composed of a

bimolecular layer of phospholipids in which 'float' proteins of various sorts. The

membrane of endoplasmic reticulum remains continuous with the membranes of

plasma membrane, nuclear membrane and Golgi apparatus. The cavity of the

endoplasmic reticulum is well developed and acts as a passage for the secretory

products. Palade (1956) has observed secretory granules in the cavity of endoplasmic

reticulum.

Sometimes, the cavity of RER is very narrow with two membranes closely

apposed and is much distended in certain cells which are actively engaged in protein

sysnthesis (e.g., acinar cells, plasma cells and goblet cells). Weibel et al. , 1969, have

calculated that the total surface of ER contained in 1ml of liver tissue is about 11

square metres, two-third of which is or rough types (i.e., RER).

Types of Endoplasmic reticulum

31

Two types of endoplasmic reticulum have been observed in same or different

types of cells which are as follows :

1. Agranular or Smooth Endoplasmic Reticulum

This type of endoplasmic reticulum possesses smooth walls because the

ribosomes are not attached with its membranes. The smooth type of endoplasmic

reticulum occurs mostly in those cells, which are involved in the metabolism of

lipids (including steroids) and glycogen. The smooth endoplasmic reticulum is

general found in adipose cells, interstitial cells, glycogen storing cells of the liver,

conduction fibres of heart, spermatocytes and leucocytes. The muscle cells are also

rich in smooth type of endoplasmic reticulum and here it is known as sarcoplasmic

reticulum. In the pigmented retinal cells it exists in the form of tightly packed

vesicles and tubes known as myeloid bodies.

Glycosomes. Although the SER forms a continuous system with RER, it has

different morphology. For example, in liver cells it consists of a tubular network that

pervades major portion of the cytoplasmic matrix. These fine tubules are present in

regions rich in glycogen and can be observed as dense particles, called glycosomes,

in the matrix. Glycosomes measure 50 to 200 mm in diameter and contain glycogen

along with enzymes involved in the synthesis of glycogen (Rybicka, 1981). Many

glycosomes attached to the membranes of SER have been observed by electron

microscopy in the liver and conduction fibre of heart.

2. Granular of Rough Endoplasmic Reticulum

The granular or rough type of endoplasmic reticulum possesses rough walls

because the ribosomes remain attached with its membranes. Ribosomes play a vital

role in the process of protein synthesis. The granular or rough type of endoplasmic

reticulum is found abundantly in those cells which are active in protein sysnthesis

such as pancreatic cells, plasma cells, goblet cells, and liver cells. The granular type

32

of endoplasmic reticulum takes basiophilic stain due to its RNA content of

ribosomes. The region of the matrix containing granular type of endoplasmic

reticulum takes basiophilic stain and is names as ergastoplasm, basiophilic bodies,

chromophilic substances or Nissl bodies by early cytologists. In RER, ribosomes are

often present as polysomes held together by mRNA and are arranged in typical,

"rosettes" of spirals. RER contains two transmembrane glycoproteins (called

ribophorins I and II of 65,000 and 64,000 dalton MW, respectively), to which are

attached the ribosomes by their 60S subunits.

endoplasmic reticulum in the intact cell was established by Palade and Siekevitz

1956.

Enzymes of the ER membranes

The membranes of the endoplasmic reticulum are found to contain many kinds

of enzymes which are needed for various important synthetic activities. Some of the

most common enzymes are found to have different transverse distribution in the ER

membranes (Table 6-1). The most important enzymes are the stearases, NADH-

cytochrome C reductase, NADH diaphorase, glucose-6-phosphotase and Mg++

activated ATPase. Certain enzymes of the endoplasmic reticulum such as nucleotide

diphosphate are involved in the biosynthesis of phospholipids, ascorbic acid,

glucuronide, steroids and hexose metabolism. The enzymes of the endoplasmic

reticulum perform the following important functions :

1. Synthesis of glycerides, e.g., triglycerides, phospholipids, glycolipids and

plasmalogens.

2. Metabolism of plasmalogens.

3. Sythesis of fatty acids.

33

4. Biosynthesis of the steroids, e.g., cholesterol biosynthesis, steroid

hydrogenation of unsaturated bonds.

5. NADPH2+O2- requiring steroid transformations : Aromatization and

hydroxylation.

6. NADPH2+O2-requireing steroid transformations : Aromatization

hydroxylation's side-chain oxidation, thio-ether oxidations, desulphuration.

7. L-ascorbic acid metabolism.

8. UDP-glucose dephosphorylation.

9. Ary1- and steroid sulphatase.

Function of Endoplasmic reticulum

The endoplasmic reticulum acts as secretory, storage, circulatory and nervous

system for the cell. performs following important functions :

A. Common Functions of Granular and Agranular Endoplasmic Reticulum

1. The endoplasmic reticulum provides and ultrastructural skeletal framework

to the cell and gives mechanical support to the colloidal cytoplasmic

matrix.

2. The exchange of molecules by the process of osmosis, diffusion and active

transport occurs through the membranes of endoplasmic reticulum. Like

plasma membrane, the ER membrane has permeases and carries.

3. The endoplasmic membranes contain many enzymes which perform

various synthetic and metabolic activities. Further the endoplasmic

reticulum provides increase surface for various enzymatic reactions.

34

4. The endoplasmic reticulum acts as an intracellular circulatory or

transporting system. Various secretory products of granular endoplasmic

reticulum are transported to various organelles as follows : Granular

ERagranular ERGolgi membranelysosomes, transport vesicles or

secretory granules. Membrane flow many also be an important mechanism

for carrying particles, molecules and ions into and out of the cells. Export

of RNA and nucleoproteins from nucleus to cytoplasm may also occur by

this type of flow (see De Robertis and De Robertis, Jr., 1987).

5. The ER membranes are found to conduct intra-cellular impulses. For

example, the sarcoplasmic reticulum transmits impulses from the surface

membrane into the deep region of the muscle fibres.

6. The ER membranes form the new nuclear envelope after each nuclear

division.

7. The sarcoplasmic reticulum plays a role in releasing calcium when the

muscle is stimulated and actively transporting calcium back into the

sarcoplasmic reticulum when the stimulation stops and the muscle must be

relaxed.

B. Functions of Smooth Endoplasmicreticulum

Smooth ER performs the following functions of the cell :

1. Synthesis of lipids. SER perform synthesis of lipids (e.g., phospholipids,

cholesterol, etc.) and lipoproteins. Studies with radioactive precursors have

indicated that the newly synthesized phospholipids are rapidly transferred

to other cellular membranes by the help of specific cytosolic enzymes,

called phospholipids exchange proteins.

35

2. Glycogenolysis and blood glucose homeostasis. This process of glycogen

synthesis (glycogenesis) occurs in the cytosol (in glycosomes). The enzyme

UDPG-glycogen transferase, which is directly involved in the synthesis of

glycogen by addition of uridine diphosphate glucose (UDPG) to primer

glycogen is bound to the glycogen particles or glycosomes.

SER is found related to glycogenolysis or breakdown of glycogen. An

enzyme, called glucose-6-phosphatase (a marker enzyme) exists as an

integral protein of the membrane of SER (e.g., liver cell). Generally, this

enzyme acts as a glycogenic phosphorhydrolase that catalyzes the release

of free glucose molecule in the lumen of SER from its phosphorylated form

in liver. Thus, this process operates to maintain homeostatic levels of

glucose in the blood for the maintenance of functions of red blood cells and

nerve tissues.

3. Sterol metabolism. The SER contains several key enzymes that catalyze

the synthesis of cholesterol which is also a precursor substance for the

biosynthesis of two types of compounds- the steroid hormones and bile

acids :

(i) Cholesterol biosynthesis. The cholesterol is synthesized from the

acetate and its entire biosynthetic pathway involve about 20 steps,

each step catalyzed by an enzyme. Out of these twenty enzymes,

eleven enzymes are bounded to SER membranes, rest nine enzymes

are the soluble enzymes located in the cytosol and mitochondria.

Examples of SER-bound enzyme include HMG-Co A reductase and

squalene synthetase (see Thorpe, 1984).

36

(ii) Bile acid synthesis. The biosynthesis of the bile acids represents a

very complex pattern of enzymes and products. Enzymes involved in

the biosynthetic pathway of bile acids are hydroxylases, mono-

oxygenases, dehydrogenases, isomerases and reductases. For

example, by the help of the enzyme cholesterol 7-hydroxylase, the

cholesterol is first converted into 7-hydroxyl cholesterol, which is

then converted into bile acids by the help of hydroxylase enzymes.

The latter reaction requires NADPH and molecular oxygen and

depends on the enzymes of Electron transport chains of SER such as

cytochrome P-450 and NADPH-cytochrome-c reductase .

(iii) Steroid hormone biosynthesis. Steroid hormones are synthesized in the

cells of various organs such as the cortex of adrenal gland, the ovaries,

the testes and the placenta. For example, cholesterol is the precursor for

both types of sex hormones-estrogen and testosterone-made in the

reproductive tissues, and the adrenocorticoids (e.g., corticosterone,

aldosterone and cortisol) formed in the adrenal glands. Many enzymes

(e.g., dehydrogenase,s isomerases and hydroxylases) are involved in the

biosynthetic pathway of steroid hormones, some of which are located in

SER membranes and some occur in the mitochondria

4. Detoxification. Protectively, the ER chemically modifies xenobiotics (toxic

materials of both endogenous and exogenous origin), making them more

hydrophilic, hence, more readily excreted. Among these materials are drugs,

aspirin (acetyl-salicylic-acid), insecticides, anaesthetics, petroleum products,

pollutant and carcinogens (i.e., inducers of cancer ; e.g., 3-4-benzophrene and

3-methyl cholantherene).

37

The enzymes involved in the detoxification of aromatic hydrocarbonds are

aryhydraoxylases. It is now know that benzophyrene (found in charcoal-

broiled meat) is not carcinogenic, but under the action of aryl hydroxylase

enzyme in the liver, it is converted into 5,6-epoxide, which is a powerful

carcinogen (see De Robertis and De Robertis, Jr., 1987)

A wide variety of drugs (e.g., Phenobarbital), when administrated to animals,

they bring about the proliferation of the ER membranes (first RER and then

SER) and /or enhanced activity of enzymes related to detoxification (Thorpoe,

1984).

C. Functions of Rough Endoplasmic Reticulum

The major function of the rough ER is the synthesis of protein. It has long

been assumed that proteins destined for secretion (i.e., export) from the cell or

proteins to be used in the synthesis of cellular membranes are synthesized on

rough DR-bound ribosomes, while cytoplasmic proteins are translated for the

most part on free ribosomes. In fact, the array of the rough endoplasmic

reticulum provides extensive surface area for the association of metabolically

active enzymes, amino acids and ribosomes. There is more efficient

functioning of these materials to synthesize proteins when oriented on a

membrane surface than when they are simply in solution, mainly because

chemical combinations between molecules can be ccomplished in specific

38

geometric patterns.

The membrane-bound ribosomes are attached with specific binding sites or

receptors of rough ER membrane by their large 60S subunit, with small or 40S

subunit sitting on top like a cap. These receptors are membrane proteins which

extend well into and possibly through the lipid bilayer. The receptor proteins

with bound ribosomes can float laterally like other membrane proteins and

may facilitate formation of the polysome and probably translation which

requires that mRNA and ribosome move with respect to each other.

39

Further, the secretory proteins, instead of passing into the cytoplasm, appear to

pass instead into the cisternae of the rough ER and are, thus, protected from

protease enzymes of cytoplasm. It is calculated that about 40 amino acid

residues long segment at the - COOH end of the nascent protein remains

protected inside the tunnel of 'free' or 'bond' ribosomes and rest of the chain,

with-NH2 end, is protected by the lumen of RER. The passage of nascent

polypeptide chain into the ER cisterna take place during translation leaving

only a small segment exposed to the cytoplasm at any one time.

Protein glycosylation. The covalent addition of sugars to the secretory proteins

(i.e., glycosylation) is one of the major biosynthetic functions of rough ER.

Most of the proteins that are isolated in the lumen of RER before being

transported to the Golgi apparatus, lysosomes, plasma membrane or

extracellular space, are glycoproteins (A notable exception is albumin). In

contrast, very few proteins in the cytosol (Cytoplasmic matrix) are

glycosylated and those that carry them have a different sugar modification.

The process of protein glycosylation in RER lumen is one of the most well

understood cell biological phenomena. During this process, a single species of

loligosaccharide (Which comprises N-acetyl-glucosamine, mannose and

glucose, containing a total of 14 sugar residues) is transferred to proteins in the

ER.

Microbodies : Structure and Types

Microbodies are spherical or oblate in form. They are bounded by a single

membrane and have an interior or matrix which is amorphous or granular Micrbodies

are most easily distinguished from other cell organelles by their content of catalase

40

enzyme. Catalase can be visualized with the electron microscope when cells are

treated with the stain DAB (i.e., 3,3'-diaminobenzidine). The product is electron

opaque and appears as dark regions in the cell where catalase is present

The technique of isolation of microbodies of plant tissues includes the

following steps : (1) Tissues are group very carefully to save microbodies from

disruption. (2) The homogenate is with differential centrifugation to obtain a fraction

of the cell homogenate which is rich in microbodies. (3) The enriched fraction is

subjected to isopycnic ultra-centrifugation on discontinuous or continuous sucrose

density gradient. Recent biochemical studies have distinguished two types of

microbodies, namely peroxisomes and glycoxysomes. These two organelles differ

both in their enzyme complement and in the type of tissue in which they are found.

Peroxisomes are found in animal cells and the leaves of higher plants. They contain

catalases and oxidases (e.g., D-amino oxidase and urate oxidase). In both they

participate in the oxidation of substrates, producing hydrogen peroxide which is

subsequently destroyed by catalase activity.

In plant cells, peroxisomes remain associated with ER. chloroplasts and

mitochondria and are involved in photorespiration. Gloxysomes occur only in plant

cells and are particularly abundant in germinating seeds which store fats as a reserve

food material. They contain enzymes of glyoxylate cycle besides the catalases and

oxidases

Peroxisomes

Peroxisomes occur in many animal cells and in a wide range of plants. They

are present in all photosynthetic cells of higher plants in etiolated leaf tissue, in

coleoptiles and hypocotyls, in tobacco stem and callus, in ripening pear fruits and

also in Euglenophyta, Protozoa, brown algae, fungi liverworts, mosses and ferns.

41

Peroxisomes are variable in size and shape, but usually appear circular in cross

section having diameter between 0.2 and 1.5m (0.2 and 0.25 m diameter in most

mammalian tissues : 0.5m diameter in rat liver cells). They have a single limiting

unit membrane of lipid and protein molecules, which encloses their granular matrix.

In some cases (e.g., in the festuciod grasses) the matrix contains numerous threads or

fibrils, while in others they are observed to contain either an amorphous nucleoid or

a dense inner core which in many species shows a regular crystalloid structure (e.g.,

tobacco leaf cell, Newcomb and Frederick, 1971). Little is known about the function

of the core, except that it is the site of the enzyme urate oxidase in rat liver

peroxisomes and much of the catalase in some plants (see Hall et al., 1974).

Recently, a possible relationship has been stressed between peroxides and free

radicals (such as superoxide anion -O2-) with the process of aging. These radicals

may act on DNA molecule to produce mutations altering the transcription into

mRNA and the translation into proteins. In addition, free radicals and peroxides can

affect the membranes by causing peroxidation of lipids and proteins. For these

reasons reducing compounds such as vitamin E or enzymes such as superoxide

dismutase could play a role in keeping the healthy state of a cell.

.

Biogenesis of Peroxisomes

At one time it was thought that the membrane 'shell' of the peroxisomes is

formed by building of the endoplasmic reticulum (ER), while the 'content' or matrix

is imported from the cytosol (cytoplasmic matrix). However, there is now evidence

suggesting that new peroxisomes always arise from pre-existing ones, being formed

by growth and fission of old organelles similar to mitochondria and chloroplasts.

Thus, peroxisomes are a collection of organelles with a constant membrane

and a variable enzymatic content. All of their proteins (both structural and

42

enzymatic) are encoded by nuclear genes and are synthesized in the cytosol

(cytoplasmic matrix) (i.e., on the free ribosomes). The proteins present in either

lumen or membrane of the peroxisome are taken up post-translationally from the

cytosol (cytoplasmic matrix) as the haeme-free monomer; the monomers are

imported into the lumen of peroxisomes, where they assemble into tetramers in the

presence of haeme./ Catalase and many peroxisomal proteins are found to have a

signal sequence (comprising of three amino acids) which is located near their

carboxyl ends and directs them to peroxisome (Gould, Keller and Subramani,1988).

Peroxisomes contain receptors exposed on their cytosolic surface to recognize the

signal on the imported proteins. All of the membrane proteins of the peroxisomes.

Including signal receptor proteins, are imported directly from the cytosol

(cytoplasmic matrix). The lipids required to make new peroxisomal membrane are

also imported from the cytosol (cytoplasmic matrix), possibly being carried by

phospholipids transfer proteins from sites of their synthesis in the DR membranes

(Affe and Kennedy, 1983).

Glyoxysomes

Glyoxysomes are found to occur in the cells of yeast, Neurospora, and oil rich

seeds of many higher plants. They resemble with peroxisomes in morphological

details, except that, their crystalloid core consists of dense rods of 6.0 m diameter.

They have enzymes for fatty acid metabolism and gluconeogenesis, i.e. conversion

of stored lipid molecules of spherosomes of germinating seeds into the molecules of

carbohydrates.

Functions

Glyoxysomes perform following biochemical activities of plants cells :

(1) Fatty acid metabolism. During germination of oily seeds, the stored lipid

molecules of spherosomes are hydrolysed by the enzyme lipase (glycerol ester

43

hydrolase) to glycerol and fatty acids. The phospholipids molecules are

hydrolysed by the enzyme phospholipase. The long chain fatty acids which are

released by the hydrolysis are then broken down by the successive removal of

two carbon or C2 fragments in the process of -oxidation.

During -oxidation process, the fatty acid is first activated by enzyme fatty

acid thiokinase to fatty acyl-CoA which is oxidized by a FAD-linked enzyme fatty

acyl-CoA dehydrogenase into-2-enoyl-CoA. Trans-2-enoyl-CoA is hydrated by an

enzyme enoyl hydratase or crotonase to produce the L-3-hydroxyacyl-CoA, which is

oxidized a NAD Linked L-3-hydratase or crotonase to produce the L-3-hydroxyacyl-

CoA, which is oxidized by a NAD linked L-3-hydroxyacyl-CoA dehydrogenase to

produce 3-Ketoacly-CoA. The 3-keto acyl-CoA looses a two carbon fragment under

the action of the enzyme thiolase to generate an acetyl-CoA and a new fatty acyl-

CoA with two less carbon atoms thatn the original. This new fatty acyl-CoA is then

recycled thought the same series of reactions until the final two molecules of acetyl-

CoA are produced.

In plant seeds -oxidation occurs in glyoxysomes (Cooper and Beevers, 1969).

But in other plant cells -oxidation occurs in glyxysomes and mitochondria. The

glyoxysomal -oxidation requires oxygen for oxidation of reduced flavorprotien

produced as a result of the fatty-acyl-CoA dehydrogenase activity. In animal cells -

oxidation occurs in mitochondria.

In plant cells, the acetyl-CoA, the product of -oxidation chain is not oxidized

by Krebs cycle, because it remains spatially separated from the enzymes of Krebs

cycle, instead of it, acetyl-CoA undergoes the glyoxylate cycle to be converted into

succinate.

(2) Glyoxylate cycle. The glyoxylate pathway occurs in glyoxysomes and it

involve some of the reactions of the Krebs cycle in which citrate is formed

44

from oxaloacetate and acetyl-CoA under the action of citrate synthetase

enzymes. The citrate is subsequently converted into isocitrate by aconitase

enzyme. The cycle then involves the enzymatic conversion of isocitrate to

glyoxylate and succinate by isocitratase enzyme :

Isocitratase

Isocitrate Glyoxylate + Succinate

The glyoxylate and another mole of acetyl-CoA form a mole of malate by

malate synthesis ;

Malate synthetase

Acetyl CoA+Glyoxylate Malate

This malate is converted to oxaloacetate by malate dehydrogenase for the

cycle to be completed. Thus, overall, the glyoxylate pathway involves :

2 Acetyl-CoA+NAD+ Succintiate + NADH+ H

+

Succinate is the end product of the glyoxysomal metabolism of fatty acid and

is not further metabolized within this organelle.

The synthesis of hexose or gluconeogenesis involves the conversion of

succinate to oxaloacetate, which presumably takes place in the mitochondria, since

the glyoxsomes do not contain the enzymes fumarase and succinic dehydrogenase.

Two molecules of oxaloacetate are formed from four molecules of acetyl-CoA

without carbon loss. This oxaloacetate is converted to phosphoenol pyruvate in the

phosphoenol pyruvate caboxykinase reaction with the loss of two molecules of CO2 :

2 Oxaloacete + 2ATP 2 Phosphoenol pyruvate + 2CO2 + 2ADP

45

MITOCHONDRIA

In cell biology, a mitochondrion (plural mitochondria) is a membrane-

enclosed organelle found in most eukaryotic cells. These organelles range

from 0.5 to 10 micrometers (μm) in diameter. Mitochondria are sometimes

described as "cellular power plants" because they generate most of the cell's

supply of adenosine triphosphate (ATP), used as a source of chemical energy.

In addition to supplying cellular energy, mitochondria are involved in a range

of other processes, such as signaling, cellular differentiation, cell death, as

well as the control of the cell cycle and cell growth. Mitochondria have been

implicated in several human diseases, including mitochondrial disorders and

cardiac dysfunction, and may play a role in the aging process. The word

mitochondrion comes from the Greek μίτος or mitos, thread + χονδρίον or

chondrion, granule.Several characteristics make mitochondria unique. The

number of mitochondria in a cell varies widely by organism and tissue type.

Many cells have only a single mitochondrion, whereas others can contain

several thousand mitochondria. The organelle is composed of compartments

that carry out specialized functions. These compartments or regions include

the outer membrane, the intermembrane space, the inner membrane, and the

cristae and matrix. Mitochondrial proteins vary depending on the tissue and

the species. In humans, 615 distinct types of proteins have been identified

from cardiac mitochondria, whereas in Murinae (rats), 940 proteins encoded

by distinct genes have been reported. The mitochondrial proteome is thought

46

to be dynamically regulated Although most of a cell's DNA is contained in the

cell nucleus, the mitochondrion has its own independent genome. Further, its

DNA shows substantial similarity to bacterial genomes.

Structure

A mitochondrion contains outer and inner membranes composed of phospholipid

bilayers and proteins.[6]

The two membranes, however, have different properties.

Because of this double-membraned organization, there are five distinct

compartments within the mitochondrion. There is the outer mitochondrial membrane,

the intermembrane space (the space between the outer and inner membranes), the

47

inner mitochondrial membrane, the cristae space (formed by infoldings of the inner

membrane), and the matrix (space within the inner membrane).

Outer membrane

The outer mitochondrial membrane, which encloses the entire organelle, has a

protein-to-phospholipid ratio similar to that of the eukaryotic plasma membrane

(about 1:1 by weight). It contains large numbers of integral proteins called porins.

These porins form channels that allow molecules 5000 Daltons or less in molecular

weight to freely diffuse from one side of the membrane to the other. Larger proteins

can enter the mitochondrion if a signaling sequence at their N-terminus binds to a

large multisubunit protein called translocase of the outer membrane, which then

actively moves them across the membrane. Disruption of the outer membrane

permits proteins in the intermembrane space to leak into the cytosol, leading to

certain cell death. The mitochondrial outer membrane can associate with the

endoplasmic reticulum (ER) membrane, in a structure called MAM (mitochondria-

associated ER-membrane). This is important in ER-mitochondria calcium signaling

and involved in the transfer of lipids between the ER and mitochondria

] Intermembrane space

The intermembrane space is the space between the outer membrane and the inner

membrane. Because the outer membrane is freely permeable to small molecules, the

concentrations of small molecules such as ions and sugars in the intermembrane

space is the same as the cytosol. However, large proteins must have a specific

signaling sequence to be transported across the outer membrane, so the protein

composition of this space is different from the protein composition of the cytosol.

One protein that is localized to the intermembrane space in this way is cytochrome c.

48

Inner membrane

The inner mitochondrial membrane contains proteins with five types of

functionsThose that perform the redox reactions of oxidative phosphorylation

1. ATP synthase, which generates ATP in the matrix

2. Specific transport proteins that regulate metabolite passage into and out of the

matrix

3. Protein import machinery.

4. Mitochondria fusion and fission protein

It contains more than 151 different polypeptides, and has a very high protein-to-

phospholipid ratio (more than 3:1 by weight, which is about 1 protein for

15 phospholipids). The inner membrane is home to around 1/5 of the total protein in

a mitochondrion. In addition, the inner membrane is rich in an unusual phospholipid,

cardiolipin. This phospholipid was originally discovered in cow hearts in 1942, and

is usually characteristic of mitochondrial and bacterial plasma membranes.

Cardiolipin contains four fatty acids rather than two and may help to make the inner

membrane impermeable. Unlike the outer membrane, the inner membrane doesn't

contain porins and is highly impermeable to all molecules. Almost all ions and

molecules require special membrane transporters to enter or exit the matrix. Proteins

are ferried into the matrix via the translocase of the inner membrane (TIM) complex

or via Oxa1. In addition, there is a membrane potential across the inner membrane

formed by the action of the enzymes of the electron transport chain.

49

The inner mitochondrial membrane is compartmentalized into numerous cristae,

which expand the surface area of the inner mitochondrial membrane, enhancing its

ability to produce ATP. For typical liver mitochondria the area of the inner

membrane is about five times greater than the outer membrane. This ratio is variable

and mitochondria from cells that have a greater demand for ATP, such as muscle

cells, contain even more cristae. These folds are studded with small round bodies

known as F1 particles or oxysomes. These are not simple random folds but rather

invaginations of the inner membrane, which can affect overall chemiosmotic

function.One recent mathematical modeling study has suggested that the optical

properties of the cristae in filamentous mitochondria may affect the generation and

propagation of light within the tissue.

Matrix

The matrix is the space enclosed by the inner membrane. It contains about 2/3 of the

total protein in a mitochondrion. The matrix is important in the production of ATP

with the aid of the ATP synthase contained in the inner membrane. The matrix

contains a highly-concentrated mixture of hundreds of enzymes, special

mitochondrial ribosomes, tRNA, and several copies of the mitochondrial DNA

genome. Of the enzymes, the major functions include oxidation of pyruvate and fatty

acids, and the citric acid cycleMitochondria have their own genetic material, and the

machinery to manufacture their own RNAs and proteins).

Organization and distribution

Mitochondria are found in nearly all eukaryotes. They vary in number and location

according to cell type. A single mitochondrion is often found in unicellular

50

organisms. Conversely, numerous mitochondria are found in human liver cells, with

about 1000–2000 mitochondria per cell making up 1/5 of the cell volume.[6]

The

mitochondria can be found nestled between myofibrils of muscle or wrapped around

the sperm flagellum.[6]

Often they form a complex 3D branching network inside the

cell with the cytoskeleton. The association with the cytoskeleton determines

mitochondrial shape, which can affect the function as well. Recent evidence suggests

vimentin, one of the components of the cytoskeleton, is critical to the association

with the cytoskeleton.

Functions

The most prominent roles of mitochondria are to produce ATP (i.e., phosphorylation

of ADP) through respiration, and to regulate cellular metabolism. The central set of

reactions involved in ATP production are collectively known as the citric acid cycle,

or the Krebs Cycle. However, the mitochondrion has many other functions in

addition to the production of ATP.

Mitochondrial DNA.

The human mitochondrial genome is a circular DNA molecule of about 16 kilobases.

It encodes 37 genes: 13 for subunits of respiratory complexes I, III, IV and V, 22 for

mitochondrial tRNA (for the 20 standard amino acids, plus an extra gene for leucine

and serine), and 2 for rRNA. One mitochondrion can contain two to ten copies of its

DNA. As in prokaryotes, there is a very high proportion of coding DNA and an

absence of repeats. Mitochondrial genes are transcribed as multigenic transcripts,

which are cleaved and polyadenylated to yield mature mRNAs. Not all proteins

necessary for mitochondrial function are encoded by the mitochondrial genome;

most are coded by genes in the cell nucleus and the corresponding proteins are

51

imported into the mitochondrion. The exact number of genes encoded by the nucleus

and the mitochondrial genome differs between species. In general, mitochondrial

genomes are circular, although exceptions have been reported In general,

mitochondrial DNA lacks introns, as is the case in the human mitochondrial

genome;] however, introns have been observed in some eukaryotic mitochondrial

DNA such as that of yeast] and protists including Dictyostelium discoideum.

In animals the mitochondrial genome is typically a single circular chromosome that

is approximately 16-kb long and has 37 genes. The genes while highly conserved

may vary in location. Curiously this pattern is not found in the human body louse

(Pediculus humanus). Instead this mitochondrial genome is arranged in 18

minicircular chromosomes each of which is 3–4 kb long and has one to three genes.

This pattern is also found in other sucking lice but not in chewing lice.

Recombination has been shown to occur between the minichromosomes. The reason

for this difference is not known.

52

1.4 COMPARISION OF PLANT AND ANIMAL CELLS

Comparision of plant and animal cells.

Animal Cell Plant Cell

Cilia: Present It is very rare

Shape: Round (irregular

shape)

Rectangular (fixed

shape)

Chloroplast: Animal cells don't have

chloroplasts

Plant cells have

chloroplasts because

they make their own

food

Vacuole:

One or more small

vacuoles (much

smaller than plant

cells).

One, large central

vacuole taking up 90%

of cell volume.

Centrioles: Present in all animal

cells

Only present in lower

plant forms.

Plastids: Absent Present

Cell wall: Absent Present

Plasma Membrane: only cell membrane cell wall and a cell

membrane

Lysosomes: Lysosomes occur in

cytoplasm.

Lysosomes usually not

evident.

Plant and animal cells have some structural differences.

53

PlantCell

Animal Cell

Comparison of structures between animal and plant cells

54

Cell walls in animal cells vs. plant cells.A notable difference between animal cells

and plant cells is that animal cells do not have a cell wall where as plant cells do.

Both plant and animal cells have plasma membranes.

Typical animal cell Typical plant cell

Organelles Nucleus

o Nucleolus (within

nucleus)

Rough endoplasmic reticulum

(ER)

Smooth ER

Ribosomes

Cytoskeleton

Golgi apparatus

Cytoplasm

Mitochondria

Vesicles

Lysosomes

Centrosome

o Centrioles

Nucleus

o Nucleolus (within

nucleus)

Rough ER

Smooth ER

Ribosomes

Cytoskeleton

Golgi apparatus (dictiosomes)

Cytoplasm

Mitochondria

Plastids and its derivatives

Vacuole(s)

Cell wall

55

CHECK YOUR PROGRESS 1

CHECK YOUR PROGRESS-1

OVERVIEW OF METABOLISM;-CATABOLISM AND ANABOLISM

During synthesis of metabolites energy is required and such constructive

reactions are called anabolism.Example photosynthesis

Co2 +h2o+Ribulose 1,5, diphospate- Rudp carbooxylase--------- 2 mol of 3 PGA

Where as reactions in which break down of certain metabolites occurs and energy is

released are called catabolism.Example Glycolysis,krebs cycle.. The process of

respiration is basically an oxidation- reduction process, where electrons are with

Note: Write your answer in the space given below.

Check your answer with the one at the end of the unit.

Fill in the blanks.

1. Prokaryotic cell shave ----------------- chromosomes.

2. An example of prokaryotic cell is -------------------.

3. Eukaryotic cells have------------------- and ------------------------------------.

4. Mitochondria are called as --------------------------------------------------- of the

cell.

5. Lysosomes are ------------------------------------- sacs of the cell.

6. Plant cell wall is made up of -----------------------------.

7. Animal cell have ----------------------- as reserve food material

8. Plastids are--------------------- in animal cells.

56

drawn from substrate (glucose) are accepted by various components of etc and

reducing powers, and leads to generation of precursor metabolites reducing power

NADPH+H+

+ATP. To recall, all living organisms respire to produce energy needed

to perform all vital activities. The energy required for biological activities is obtained

from organic compounds available in food. Plants synthesize their own food through

photosynthesis.

Defination : ― Respiration is a process by which organic food materials such as

sugar, fats, etc get successively oxidized to produce CO2, H2O and energy.‖

C6H12O6 + 6O2 6CO2 + 6H2O + 673Kcal energy

The overall reaction of cellular repiration is given as

C6H12O6 + 6O2 + 38Adp +38iP 6CO2 + 6H2O + 38ATP

AN OVERVIEW OF RESPIRATION.

a) You must bear a clear understanding in mind that both photosynthesis and

respiration involves gaseous exchange but light reaction of photosynthesis

requires sunlight whereas respiration occurs all the time.

O2 utilized in the process &Co2 is released .

b) The sites of respiration are cytoplasms and mitochondria. The organic

compounds are broken down inside the cells by oxidation process, known as

cellular respiration. The energy released is stored in pyrophosphate bonds of

ATP.

57

ADP + H3PO4 ATP(ADP˜P)

Energy stored in ATP is utilized for carying out different cellular and biological

activites because of this, energy is called energy currency of the cell.

c) The overall reaction is as follows:

C6H12O6 + 6O2 + 38Adp +38iP 6CO2 + 6H2O + 38ATP

The main features of respiration are:

Oxidation of organic compounds occurs in under aerobic conditions

Complete oxidation occurs

End products are CO2 & H2O

Higher amount of (673 Kcal )energy is liberated out

Process occurs in cytoplasm and mitochondria

Chlorophyll pigment is not essential

Various respiratory substance are: glucose, fructose, fats, protein, etc.

The ratio of volume of CO2 released to the volume of O2 absorbed during

respiration is called respiratory ratio or R.Q.

Volume of CO2 released

R.Q. =

Volume of O2 absorbed

To develop a clear understanding of the process let us understand the mechanism of

respiration

MECHANISM OF RESPIRATION

Cellular respiration is a complicated process which is completed in many steps. for

every step, a particular enzyme is required which works in a sequential manner one

after the another.

58

it is completed in 3 steps:

a) Glycolysis / EMP pathway

b) Oxidation of pyruvic acid

c) ETC & oxidative phosphorylation

a. GLYCOLYSIS/ EMP PATHWAY

Greek, glucose – sugar, lysis – dissolution. If I say that glycolysis is a fermentive

pathway would you agree?

Reasons to support my statement are:

a) It does not involves O2 intake

b) ATP generated is through substrate level phosphorylation.

c) Organic compound donates electrons and organic compound accepts

it.

This process was discovered by three German scientists Embden, meyerhof and

Parnas. On their name the pathway is also called EMP pathway.

All the reactions of glycolysis take place in the cytoplasm and

through the glycolysis glucose is oxidized into pyruvic acid in presence of many

enzymes present in the cytoplasm. Thus the process of sequential oxidation of

glucose into pyruvic acid is known as glycolysis.

59

β- OXIDATION OF PYRUVIC ACID OR KREBS CYCLE

All the chemical reaction of Kreb‘s cycle can be summarized in following steps:

1. Aerobic oxidation of P.A

2. Condensation of Acetyl-CoA with oxalo-acetic acid

3. Isomerisation of citric acid into isocitric acid ,( (a) dehydration and b)

hydration)

60

4. Oxidative decarboxylation of isocitric acid ((a) dehydration and b)

decarboxylation)

5. Oxidative decarboxylation of α-Keto glutaric acid.

6. Conversion of succinyl CoA into succinic acid.

7. Dehydrogenation of succinic acid into fumaric acid

8. Hydration of fumaric acid into malic acid

9. Dehydrogenation of malic acid in OAA.

Overall reaction of respiration is:

Glycolysis + Kreb‘s cycle = Glucose + 4ADP + 4H3PO4 + 8NAD+ + NADP

+ +2FAD

6CO2 + 4 ATP + 8NADH + 10H+ +2NADPH + 2FADH2

Thus as a result of oxidation of pyruvic acid, one molecule of CO2 in oxidative

decarboxylation and two molecules of CO2 in Kreb‘s cycle are liberated. The total

number of CO2 evolved becomes 3 which indicates that 3 carbon pyruvic acid has

been completely oxidized in glycolysis.

Because two molecules of P.A. which are formed by one molecule of glucose in

glycolysis, enter into Kreb‘s cycle for oxidation, a total of 6CO2 molecule will be

evolved.

2PA * 3CO2 = 6CO2

All the NADH2 and FADH2 are oxidized to NAD and FAD through a chain of

reaction c/a etc.in this process ATP molecules are released (1NADH2 = 3ATP,

1FADH2 = 2ATP). In the process of Kreb‘s cycle 8 molecules of NADH2 =24ATP ,

2FADH2 = 4 ATP and two molecules of ATP are synthesized from 2GTP.

61

c ELECTRON TRANSPORT SYSTEM AND OXIDATIVE

PHOSPHORYLATION

Electron Transport System

During repiration simple carbohydrates and intermediate compounds like

acid and malie acid are oxidized. Each oxidative step involves release of a pair of

hydrogen atoms which dissociates into two protons and two electrons.

2H 2H+ + 2e

-

These protons and electrons are accepted by various hydrogen acceptors like

NAD,NADP, FAD etc. After accepting hydrogen atoms these acceptors get reduced

to produce NADH2, NADPH2 and FADH2. The pairs of hydrogen atoms released a

series of coenzymes and cytochromes which form electron transport system, before

reacting with O2 to form H2O.

½ O + 2H+ + 2e

- H2O

2NADH + O2 + 2H+ 2NAD

++ 2H2O

As you know that H ions and electrons removed from the respiratory substrate

during oxidation do not directly react with oxygen. Instead they reduce acceptor

molecules NAD and FAD to NADH2 and FADH2. These molecules then transfertheir

electron to a system of electron acceptors and transfer molecules. The proteins of the

inner mitochondrial membrane act as electron transporting enzymes. They are

arranged in an ordered manner in the membrane and function in a specific sequence.

This assembly of electron transport enzymes is known as mitochondrial respiratory

62

chain or the electron transport chain. Specific enzymes of this chain receive electrons

from reduced prosthetic groups, NADH2 or FADH2 produced by glycolysis and the

TCA cycle. The electrons are then transported successively from enzyme to enzyme,

down a descending ‗stairway‘ of energy yielding reactions. This process takes place

in mitochondrial cristae which contain all the components of E,T.S.

Components of electron transport system: the electron transport system is made

up of following enzymes and proteins:

1. Nicotinamide adenine dinucleotide (NAD).

2. Flavoproteins (FAD and FMN).

3. Fe-S protein complex.

4. Co-enzyme Q or ubiquinone.

5. Cytochome-b

6. Cytochrome-c1.

7. Cytochrome-c

8. Cytochrome-a

9. Cytochrome-a3.

All the above enzymes are found in F1 particles of mitochondria.

Mechanism of action of electron transport system: During respiration electron

pairs liberated from respiratory compounds are accepted by coenzymes like NAD

or NADP and FMN etc. The transfer of electrons in all compounds except

succinic acid takes place first in NAD+ or NADP

+ and later on in FAD. The

transfer of electgrons from succinic acid takes place diretly to the FAD and not

through NAD+ or NADP

+. Due to this reason only two molecules of ATP are

63

formed in the formation of fumaric acid from succinic acid whereas in case of

other compounds 3 ATP molecules are produced because these cases the

electrons are first picked up by NAD.

Different Steps of E.T.S. are as follows:

1. Hydrogne pairs released from different substrates of Krebs cycle except

succinic acid reacts with NAD+. The electrons and proton are transferred to

NAD causing its reduction and one proton is released in the medium.

2H 2H+ + 2e

-

(protons) (electrons)

NAD + 2H+ + 2e

- NADH + H

+

(reduced) (ion pool)

2. Now, 2e- and one H

+ are transferred from NADH to FAD causing oxidation

of NADH to NAD and reduction of FMN into FMNH2. One H+ is picked

up from hydrogen ion pool to complete this reaction.

The free energy released at this step is stored during oxidative phophorylation

and one molecule of ATP is generated fronm ADP and inorganic phosphate.

The hydrogen pair from succinic acid is first transferred to FAD to form

FADH2. The FADH2 transfers electrons to coenzyme Q throught Fe-S and CoQ. The

electrons pass to cytochromes Cyt-b, Cyt-c1, Cyt-c, Cyt-a, Cyt-a3 and then to oxygen

atoms. Oxygen atom accepts those electrons and reacts with hydrogen ions of the

matrix to form water.

O2 + 4e- 2(O

--)

2(O--) + 4e

+ 2H2O

64

Oxygen is thus the terminal electron acceptor of the mitochondrial respiratory

chain.

At each step of electron acceptor has a higher electron affinity than the electron

donor from which it receives the electron. The energy from such electron transport is

utilized in transporting protons from the matrix across the inner membrane to its

outer side. This creates a higher proton concentration outside the inner membrane

than in the matrix. The difference in proton concentration across the inner membrane

is called proton gradient.

The reduction of various cytochromes requires only electrons and no protons.

Each cytochromes possesses an iron elements in the centre which functions for

accepting (Fe3+

Fe2+

) or donating (Fe2+

Fe3+

) When a cytochrome accepts electrons, it

is reduced and if it donates electrons, it is oxidized.

Oxidative Phosphorylation

In all living beings ATP generated during oxidative breakdown of complex food

products. This process of synthesis of ATP molecules from ADP and inorganic

phosphate by electron transport system of aerobic respiration called as oxidative

phosphorylation.

ADP + iP O

2 ATP

E.T. Chain

The process of oxidative phosphorylation takes place in mitochondrial crests through

electron transport chain.

Due to high proton concentration outside the inner membrane, protons return

to the matrix down the proton gradient. Just as a flow of water from a higher to lower

65

level can be utilized to turn a water-wheel or a hydroelectric turbine, the energy

released by the flow of protons down the gradient is utilized in synthesizing ATP.

The return of proton occurs through the inner membrane particles. In the F0-F1

complex the F1 head piece functions as ATP synthetase. The latter synthesizes ATP

from ADP and inorganic phosphate using the energy from the proton gradient.

Transport of two electrons from NADH2 by the electron transport chain

simultaneously transfers three pairs of protons to the outer compartment. One high

energy ATP bond is produced per pair of protons returning to the matrix through the

inner membrane particles. Therefore, oxidative phosphorylation produces three ATP

molecules per molecules of NADH2 oxidized. Since FADH2 donates its electrons

further down the chain. Its oxidation can only produce two ATP molecules.

During oxidative phosphorylation ATP molecules are produced during following

steps:

I. When NADH2 is oxidized to NAD by reacting with FAD.

II. When the electron transfer from cytochrome-b to cytochrome-c1.

III. When the electron transfer from cytochrome-a to cytochrome-a3.

Now it is clear that oxidation of one molecule of reduced NADH2 or NADPH2

results in the formation of 3 molecules of ATP while oxidation of FADH2 leads to

the formation of 2 molecules of ATP.

66

1.6 ATP THE BIOLOGICAL ENERGY CURRENCY

Defination : ― Respiration is a process by which organic food materials such as

sugar, fats, etc get successively oxidized to produce CO2, H2O and energy.‖

C6H12O6 + 6O2 6CO2 + 6H2O + 673Kcal energy

The overall reaction of cellular repiration is given as

C6H12O6 + 6O2 + 38Adp +38iP 6CO2 + 6H2O + 38ATP

Higher amount of (673 Kcal )energy is liberated out Objective:

The main aim of this unit is to develop an understanding of the process of

respiration. After learning this unit you should be able to

Differentiate between various types of (respiration, fermentation) fueling

reaction.

Understand the significance of respiration in

a) Generation of precursors

b) Generation of reducing power

c) Generation of ATP

Realize the role and significance of various enzymes involved in the process.

Understand the existence of alternative oxidation pathways

The applications of fermentation and the basic difference between the process

of aerobic respiration , anaerobic respiration & fermentation.

Role of ATP :

Adenosine - P ~ P ~ P + H2O adenosine - P~P + P 4° = -

7.8Kcal

67

Adenosine - P ~ P + H2O adenosine ~P + P 4° = -

7.3Kcal

Adenosine ~ P + H2O adenosine + P 4° = -

3.4Kcal

High energy compounds other than ATP

Compound cause action in Priosyn.of:

GTP Protein(ribosome function)

CTP Phospholipids

UTP Peptidoglycan layer of bacterial

wall

Dcoxythymidine~ P~P~P lipopolysaccarid layer of bacterial

wall

dTTTP

Acyl~SCoA Fatty acids

1.7 ORIGIN OF LIFE UNIQUE PROPERTIIES OF CARBON,CHEMICAL

EVOLUTION AND RISE OF LIVING SYSTEMS

The origin of cells has to do with the origin of life, which began the history of

life on Earth.

Origin of the first cell

There are several theories about the origin of small molecules that could lead to life

in an early Earth. One is that they came from meteorites Another is that they were

created at deep-sea vents. A third is that they were synthesized by lightning in a

reducing atmosphere); although it is not clear if Earth had such an atmosphere. There

are essentially no experimental data defining what the first self-replicating forms

68

were. RNA is generally assumed to be the earliest self-replicating molecule, as it is

capable of both storing genetic information and catalyzing chemical reactions (see

RNA world hypothesis). But some other entity with the potential to self-replicate

could have preceded RNA, like clay or peptide nucleic acid.

Cells emerged at least 4.0–4.3 billion years ago. The current belief is that these cells

were heterotrophs. An important characteristic of cells is the cell membrane,

composed of a bilayer of lipids. The early cell membranes were probably more

simple and permeable than modern ones, with only a single fatty acid chain per lipid.

Lipids are known to spontaneously form bilayered vesicles in water, and could have

preceded RNA. But the first cell membranes could also have been produced by

catalytic RNA, or even have required structural proteins before they could form.

Origin of eukaryotic cells

The eukaryotic cell seems to have evolved from a symbiotic community of

prokaryotic cells. DNA-bearing organelles like the mitochondria and the chloroplasts

are almost certainly what remains of ancient symbiotic oxygen-breathing

proteobacteria and cyanobacteria, respectively, where the rest of the cell seems to be

derived from an ancestral archaean prokaryote cell – a theory termed the

endosymbiotic theory.

There is still considerable debate about whether organelles like the hydrogenosome

predated the origin of mitochondria, or viceversa: see the hydrogen hypothesis for

the origin of eukaryotic cells.

Sex, as the stereotyped choreography of meiosis and syngamy that persists in nearly

all extant eukaryotes, may have played a role in the transition from prokaryotes to

eukaryotes. An 'origin of sex as vaccination' theory suggests that the eukaryote

69

genome accreted from prokaryan parasite genomes in numerous rounds of lateral

gene transfer. Sex-as-syngamy (fusion sex) arose when infected hosts began

swapping nuclearized genomes containing co-evolved, vertically transmitted

symbionts that conveyed protection against horizontal infection by more virulent

symbionts.

1.8 INTRODUCTION TO BIOMOLECULES

The living organisms are composed of molecules which are intrinsically

inanimate.These molecules confer a remarkable combination of characteristics called

life.Many of he most important molecules in biological systems are polymers,that is

large molecules made up of smaller subunits joined together by covalent bonds,and

in some cases in a specific order. Most of the molecular constituents of living

systems are composed of C-atoms joined with other carbon atoms and with hydrogen

,oxygen or nitrogen.Thus form a great variety of molecules such as aminoacids

,monosaccharides,nucleotides and fattyacids.These are called micromolecules.These

micromolecules serve as monomeric subunits of proteins,nucleic acids,

polysaccharides and lipids respectively which are designated as

macromolecules.These macromolecules serve more than one functions in living

cells and in all living organisms

CARBOHYDRATES

Carbohydrates are madeup of just threedifferent elements,carbon,hydrogen,and

oxygen.the simplest carbohydrates are monosaccharides,,have the general formula

(ch2o)n.they are classed as either aldoses or ketoses.example glucose.A disaccharide

70

is formed when two monosaccarides join together with a concomitant loss of water

molecules.Further monosaccaharides can be added,giving chains of three, four ,five

or more units.these are termed oligosaccarides and chain with many units are

polysaccharides.The chemical bond joinig the monosaccharide units together is

called a glycosidic linkage.

Biologically important molecules such as starch ,cellulose and glycogenare all

polysaccharides.serve as major components of the cell walls of bacteria and of the

soft cell coats in animal tissues.

The carbohydrates, or saccharides, are most simply

defined as polyhydroxy aldehydes or ketones and their

derivatives. Many have the empirical formula (CH2O)n,

which originally suggested they were ―hydrates‖ of

carbon. Monosaccharides, also called simple sugars,

consist of a single polyhydroxy aldehyde or ketone unit.

The most abundant monosaccharide is the six-carbon

D-glucose; it is the parent monosaccharide from which

most others are derived. D-Glucose is the major fuel for

most organisms and the basic building block of the

most abundant polysaccharides, such as starch and

cellulose.

Oligosaccharides (Greek oligo, ―few‖) contain from two

to ten monosaccharide units joined in glycosidic

linkage. Polysaccharides contain many monosaccharide

units joined in long linear or branched chains. Most

polysaccharides contain recurring monosaccharide units of only a single kind or two

alternating kinds.

71

Polysaccharides have two major biological functions, as a storage form of fuel and as

structural elements. In the biosphere there is probably more carbohydrate than all

other organic matter combined, thanks largely to the abundance in the plant world of

two polymers of D-glucose, starch and cellulose. Starch is the chief form of fuel

storage in most plants, whereas cellulose is the main extracellular structural

component of the rigid cell walls and the fibrous and woody tissues of plants.

Glycogen, which resembles starch in structure, is the chief storage carbohydrate in

animals. Others polysaccharides

PROTEINS

Proteins are the most abundant molecules in cells, consisting 50 percent or more of

their dry weight. They are found in every part of every cell, since they are

fundamental in all aspects of cell structure and function. There are many different

kinds of proteins, each specialized for a different biological function. Moreover,

most of the genetic information is expressed by proteins. The structure of protein

molecules and its relationship to their biological function and activity are central

problems in biochemistry today.

Proteins consist of long chains, in which amino acids occur in specific linear

sequences. Yet we know that in each type of protein the polypeptide chain is folded

into a specific three-dimensional conformation, which is required for its specific

biological function and activity. How is the linear, or one-dimensional, information

inherent in the amino acid sequence of polypeptide chains translated into the three-

dimensional conformation of native protein molecules?

The answer to this question comes from some of the most significant advances in

modern biological research. These discoveries, made possible by the application of

72

physical-chemical measurements to pure proteins, have illuminated the function and

comparative biology of proteins.

In this chapter, we examine various aspects of the primary structure of proteins,

which we have defined as the covalent backbone structure of polypeptide chains,

including the sequence of amino acid residues. We begin by considering the

properties of simple peptides. Then we examine three major aspects: (1) the

determination of amino acid sequence in polypeptide chains, (2) the significance of

variations in the amino acid sequences of different proteins in different species, and

(3) the laboratory synthesis of polypeptide chains.(4) structure of proteins.

LIPIDS

Lipids are esters of higher fatty acids. Lipids are water-insoluble organic

biomolecules that can be extracted from cells and tissues by nonpolar solvents, e.g.,

choloroform, ether, or benzene. There are several different families or classes of

lipids but all derive their distinctive properties from the hydrocarbon nature of a

major portion of their structure. (1) as structural components of membranes, (2) as

storage and trasport forms of metabolic fuel, (3) as a protective coating on the

surface of many organisms, and (4) as cell-surface components concerned in cell

recognition, species specificity, and tissue immunity. Some substances classified

among the lipids have intense biological activity; they include some of the vitamins

and hormones.

Although lipids are a distinct class of biomolecules, we shall see that they often

occur combined, either covalently or through weak bonds, with members of other

classes of bio-molecules to yield hybrid molecules such as glycolipids, lipoproteins,

73

which contain both lipids and proteins. In such biomolecules the distinctive chemical

and physical properties of their components are blended to fill specialized biological

functions.

NUCLEIC ACIDS

DEOXYRIBONUCLEIC ACID - DNA AND RIBONUCLEIC ACID RNA

Occurrence

DNA is found in the cells of all living organisms except plant viruses, where RNA

forms the genetic material and DNA is absent. In bacteriophages and viruses there is

a single molecule of DNA, which remains coiled and is enclosed in the protein coat.

In bacteria, mitochondria and plastids of eukaryotic cells DNA is circular and lies

naked in the cytoplasm. In the nuclei of eukaryotic cells DNA occurs in the form

long spirally coiled and unbranched threads. The number of DNA molecules is

equivalent to the number of chromosomes per cell. In them DNA is found in

combination with proteins forming nucleoproteins or the chromatin material and is

enclosed in the nucleus.

The nucleic acids are of considerable importance in biological systems.Two types of

nucleic acids are found in the cells of all living organisms. These are:

1. Deoxyribonucleic acid - DNA

2. Ribonucleic acid - RNA

The nucleic acid was first isolated by Friedrich Miescher in 1868 from the nuclei of

pus cells and was named nuclein. At that time its biological significance was barely

understood. The name nucleic acid was given to it after knowing its acidic property.

74

They are of two types; (1) Ribose nucleic acid, and(2) Deoxyribose nucleic acid .

The basic chemical subunits of the nucleic acids are nucleotides. The nucleotides are

made up of three components: (i) A heterocyclic ring containing nitrogen, known as

a nitrogenous base, (ii) a five carbon pentose sugar, and (iii) A phosphate group.

The bases found in nucleic acid are of two kinds- purines and pyrimidines.Adenine

and guanine are purine and cytosine, uracil and thymine are pyrimidinebases.The

nucleotides found in nucleic acids are much fewer in number than the α-amino acids.

DNA is found in almost all the cells as a major component of chromosomes of the

nucleus.. Certain viruses, including many of the bacterial viruses or bacteriophages,

are DNA-protein particles. Mostly the plant viruses are RNA-protein particles.

Ribose nucleic acid (RNA) is also of common occurrence in plants as well as

animals. It is of three types- (i)ribosomal RNA (r-RNA); (ii) soluble RNA or transfer

RNA (t-RNA) and (iii) messenger RNA (m-RNA). Ribosomal-RNA is found in

small sub-cellular particles, the ribosomes. RNAs with sendimentation Coefficient

value, 5S, 16S and 23S have been reported from 70S ribosomes, while 18S, 28S,

5.8S and 5S r-RNAs have been reported from 80S ribosomes. T-RNA is found in

free from in the cytoplasm. M-RNA is found in small quantities in association with

ribosomes.

THE CHEMICAL BASIS OF HEREDITY

DNAs are present mainly in the nucleus (in chromosomes) of the cell so they are the

carrier of gnetic information because the DNA molecule can produce a copy of itself

each going to one cell i.e. the parent DNA molecule gives rise to two identical

daughter molecules each going to one cell and thus each daughter cell receives

exactly the same complement of DNA ( both qualitatively and quantitatively as the

parent cell.

75

DNA as the bearer of genetic information in the cell is strongly supported by the

Watson-Crick structure for this compound which explains beautifully the

phenomenon of replication (and hence genetic continuity) of DNA.

This phenomenon of DNA replication can be explained as below: the double helix of

DNA separates into two strands: the individual strands combine in sequence with

their complementary free nucleotides (present in nuclear sap) through specific

hydrogen bonding ( Adenine….Thymine, Guanine…Cytisine) and now phosphate

ester linkages are formed between two nucleotides by the enzyme catalase.

Thus DNA in animals, plants, bacterial cells, and some viruses maintain the genetic

continuity. The direct evidence in favour of the genetic role of DNA is derived from

the process of bacterial transformation. If an extract of the strain of pneumococcus,

possessing capsules having specific polysaccharides, is added to the culture of strain

of pneumococcus not having the above mentained capsules, the latter is transformed

to the former. The active agent (transforming factor) is a DNA molecule which

endows the bacterial cell with the capacity for synthesizing an enzyme or enzyme

system not present previously in non-encapsulated strain. This enzyme in turn

catayzes the formation of the specific capsular polysaccharide.

76

1.9 BUILDING BLOCKS OF BIOMACROMOLECULES

The molecular constituents of living systems are composed of C-atoms joined with

other carbon atoms and with hydrogen ,oxygen or nitrogen.They form a great variety

of molecules suc nucleic aci ds h as aminoacids, monosaccharides, nucleotides and

fattyacids.These are called micromolecules.These micromolecules serve as

monomeric subunits of proteins, polysacchrides, nucleic acids and lipids.

A. MICROMOLECULES OF CARBOHYDRATES

Monosaccharides have the empirical formula (CH2O)n, where n=3 or some larger

number. The carbon skeleton of the common monosaccharides is unbranched and

each carbon atom except one

contains a hydroxyl group; at

the remaining carbon there is

a carbonyl oxygen, which, as

we shall see, is often

combined in an acetal or ketal

linkage. If the carbonyl

groups is at the end of the

chain, the monosaccharide is

an aldehyde derivative and

called an aldose; if it is at any

other position, the

monosaccharides is a ketone

derivative and called ketose.

Fig. 2

77

The simplest monosaccharides are the three carbon trioses glyceraldehydes and

dihydrosyacetone. Glyceraldehyde is an aldotriose; dihydroxyacetone is a ketotriose.

Also among the monosaccharides are the tetroses (four carbons), pentoses (five

carbons), hexoses (six carbons), heptoses (seven carbons), and octoses (eight

carbons). Each exists in two series, i.e., aldotetroses and ketotetroses, aldopentoses

and ketopentoses, aldohexoses and ketohexoses, etc. The structures of D aldoses and

D ketoses are shown. In both classes of monosaccharides the hexoses are by far the

most abundant. However, aldopentoses are important components of nucleic acids

and various polysaccharides; derivatives of trioses and heptoses are important

intermediates in carbohydrate metabolism. All the simple monosaccharides are white

crystalline solids that are free soluble in water but insoluble in nonpolar solvents.

Most have sweet taste.

CONFIGURATION OF MONOSACCHARIDES & THEIR FAIMILY

First, we must clarify two terms often confused. Configuration denotes the

arrangement in space of substituent groups in stereoisomers such structures cannot

be inter-converted without breaking one or more covalent bonds. Conformation

refers to the spatial arrangement of substituent groups that are free to assume many

different positions, without breaking bonds, because of rotation about the single

bonds in the molecule. In the hydrocarbon ethane, for example, one might expect

complete freedom of rotation around the C-C single bond to yield an infinite number

of conformations of the molecule. However, the staggered conformation is more

stable than all others and thus predominates, whereas the eclipsed form is least

stable.

78

All the monosaccharides expect

dihydroxyacetone contain one or

more asymmetric carbon atoms and

thus are chiral molecules.

Glyceraldehyde contains only one

asymmetric carbon atom and

therefore can exist as two different

stereoisomers (Figure- 4). It will be

recalled that C- and L-

Glyceraldehyde are the reference, or

parent, compounds for designating

the absolute configuration of all

stereoisomeric compounds.

Aldotetroses have two asymmetric

carbon atoms and aldopentoses have

three. The aldohexoses have four

asymmetric carbon atoms and thus

exist in the form of 2n = 2

4 = 16

different stereoisomers, 8 of which

are shown in Figure- 2. As expected, the monosaccharides with asymmetric carbon

atoms are optically active. For example, the usual form of glucose found in nature in

20 = +52.7

0), and the usual form of fructose is levorotatory ([α]

20

= -92.40), but both are members of the D series since their absolute configurations

are related to D-glyceraldehyde. For sugars having two or more asymmetric carbon

atoms, the convention has been adopted that the prefixes D and L refer to the

asymmetric carbon atoms farthest removed from carbonyl carbon atom.

D

D

79

The stereoisomers of glyceraldehyde, showing projection formulas (top) and

perspective formulas (bottom).

Shows the projection formulas of the D aldoses. All have the same configuration at

the asymmetric carbon atom farthest from the carbonyl carbon, but because most

have two or more asymmetric carbon atoms, a

number of isomeric D aldoses exist, most

important biologically being D-glyceraldehyde.

D-ribose, D-glucose, D-mannose, and D-

galactose.

Figure-4 shows the projection formulas of the

D ketoses; all share the same configuration at

the asymmetric carbon atom farthest from the

carbonyl group. Ketoses are sometimes designated by inserting ul into the name of

the corresponding aldose; e.g., D-ribulose is

the ketopentose corresponding to the

aldopentose D-ribose. The most important

ketoses biologically and dihydroxyacetone. D-

ribulose, and D-fructose.

Aldoses and ketoses of the L series are mirror images of their D counterparts. L

sugars are found in nature, but they are not so abundant as D sugars. Among the

most important are L-fucose, L-rhamnose , and L-sorbose.

Two sugars differing only in the configuration around one specific carbon atom are

called epimers of each other. Thus, D-glucose and D-mannose are epimers with

respect to carbon atom 2, and D-glucose and D-galactose are epimers with respect to

carbon atom 4.

80

B. . MICROMOLECULES OF AMINOACIDS

A polypeptide is an unbranched chain of amino acids.

Standard amino acids

Amino acids are the structural units that make up proteins. They join together to

form short polymer chains called peptides or longer chains called either polypeptides

or proteins. These polymers are linear and unbranched, with each amino acid within

the chain attached to two neighboring amino acids. The process of making proteins is

called translation and involves the step-by-step addition of amino acids to a growing

protein chain by a ribozyme that is called a ribosome The order in which the amino

acids are added is read through the genetic code from an mRNA template, which is a

RNA copy of one of the organism's genes.

Twenty-two amino acids are naturally incorporated into polypeptides and are called

proteinogenic or natural amino acids. Of these, 20 are encoded by the universal

genetic code. The remaining 2, selenocysteine and pyrrolysine, are incorporated into

proteins by unique synthetic mechanisms. Selenocysteine is incorporated when the

81

mRNA being translated includes a SECIS element, which causes the UGA codon to

encode selenocysteine instead of a stop codon. Pyrrolysine is used by some

methanogenic archaea in enzymes that they use to produce methane. It is coded for

with the codon UAG, which is normally a stop codon in other organisms. This UAG

codon is followed by a PYLIS downstream sequence.

Non-standard amino acids

Aside from the 22 standard amino acids, there are many other amino acids that are

called non-proteinogenic or non-standard. Those either are not found in proteins (for

example carnitine, GABA), or are not produced directly and in isolation by standard

cellular machinery (for example, hydroxyproline and selenomethionine).

Non-standard amino acids that are found in proteins are formed by post-translational

modification, which is modification after translation during protein synthesis. These

modifications are often essential for the function or regulation of a protein; for

example, the carboxylation of glutamate allows for better binding of calcium cations,

and the hydroxylation of proline is critical for maintaining connective tissues.

Another example is the formation of hypusine in the translation initiation factor

EIF5A, through modification of a lysine residue.[26]

Such modifications can also

determine the localization of the protein, e.g., the addition of long hydrophobic

groups can cause a protein to bind to a phospholipid membrane.

82

β-alanine and its α-alanine isomer

Some nonstandard amino acids are not found in proteins. Examples include

lanthionine, 2-aminoisobutyric acid, dehydroalanine, and the neurotransmitter

gamma-aminobutyric acid. Nonstandard amino acids often occur as intermediates in

the metabolic pathways for standard amino acids — for example, ornithine and

citrulline occur in the urea cycle, part of amino acid catabolism (see below)[ A rare

exception to the dominance of α-amino acids in biology is the β-amino acid beta

alanine (3-aminopropanoic acid), which is used in plants and microorganisms in the

synthesis of pantothenic acid (vitamin B5), a component of coenzyme A.

Essential Nonessential

Isoleucine Alanine

Leucine Asparagine

Lysine Aspartic acid

Methionine Cysteine*

Phenylalanine Glutamic acid

Threonine Glutamine*

Tryptophan Glycine*

Valine Proline*

Selenocysteine*

Serine*

Tyrosine*

Arginine*

83

Histidine*

Ornithine*

Taurine*

The first few amino acids were discovered in the early 19th century. In 1806, the

French chemists Louis-Nicolas Vauquelin and Pierre Jean Robiquet isolated a

compound in asparagus that proved to be asparagine, the first amino acid to be

discovered. Another amino acid that was discovered in the early 19th century was

cystine, in 1810, although its monomer, cysteine, was discovered much later, in

1884. Glycine and leucine were also discovered around this time, in 1820.

General structure

Lysine

The carbon atom next to the carboxyl group is called the α–carbon and amino acids

with a side-chain bonded to this carbon are referred to as alpha amino acids. These

84

are the most common form found in nature. In the alpha amino acids, the α–carbon is

a chiral carbon atom, with the exception of glycine. In amino acids that have a

carbon chain attached to the α–carbon (such as lysine, shown to the right) the

carbons are labeled in order as α, β, γ, δ, and so on. In some amino acids, the amine

group is attached to the β or γ-carbon, and these are therefore referred to as beta or

gamma amino acids.Amino acids are usually classified by the properties of their

side-chain into four groups. The side-chain can make an amino acid a weak acid or a

weak base, and a hydrophile if the side-chain is polar or a hydrophobe if it is

nonpolar..

Isomerism

Of the standard α-amino acids, all but glycine can exist in either of two optical

isomers, called L or D amino acids, which are mirror images of each other (see also

Chirality). While L-amino acids represent all of the amino acids found in proteins

during translation in the ribosome, D-amino acids are found in some proteins

produced by enzyme posttranslational modifications after translation and

translocation to the endoplasmic reticulum, as in exotic sea-dwelling organisms such

as cone snails. They are also abundant components of the peptidoglycan cell walls of

bacteria, and D-serine may act as a neurotransmitter in the brain The L and D

convention for amino acid configuration refers not to the optical activity of the

amino acid itself, but rather to the optical activity of the isomer of glyceraldehyde

from which that amino acid can, in theory, be synthesized (D-glyceraldehyde is

dextrorotary; L-glyceraldehyde is levorotary). In alternative fashion, the (S) and (R)

designators are used to indicate the absolute stereochemistry. Almost all of the amino

acids in proteins are (S) at the α carbon, with cysteine being (R) and glycine non-

chiral. Cysteine is unusual since it has a sulfur atom at the second position in its

side-chain, which has a larger atomic mass than the groups attached to the first

85

carbon, which is attached to the α-carbon in the other standard amino acids, thus the

(R) instead of (S).

An amino acid in its (1) unionized and (2) zwitterionic forms

Zwitterions

The amine and carboxylic acid functional groups found in amino acids allow them to

have amphiprotic properties. Carboxylic acid groups (-CO2H) can be deprotonated to

become negative carboxylates (-CO2- ), and α-amino groups (NH2-) can be

protonated to become positive α-ammonium groups (+NH3-). At pH values greater

than the pKa of the carboxylic acid group (mean for the 20 common amino acids is

about 2.2, see the table of amino acid structures above), the negative carboxylate ion

predominates. At pH values lower than the pKa of the α-ammonium group (mean for

the 20 common α-amino acids is about 9.4), the nitrogen is predominantly protonated

as a positively charged α-ammonium group. Thus, at pH between 2.2 and 9.4, the

predominant form adopted by α-amino acids contains a negative carboxylate and a

positive α-ammonium group, as shown in structure (2) on the right, so has net zero

charge. This molecular state is known as a zwitterion, from the German Zwitter

meaning hermaphrodite or hybrid. Below pH 2.2, the predominant form will have a

neutral carboxylic acid group and a positive α-ammonium ion (net charge +1), and

above pH 9.4, a negative carboxylate and neutral α-amino group (net charge -1). The

86

fully neutral form (structure (1) on the right) is a very minor species in aqueous

solution throughout the pH range (less than 1 part in 107). Amino acids also exist as

zwitterions in the solid phase, and crystallize with salt-like properties unlike typical

organic acids or amines.

Isoelectric point

At pH values between the two pKa values, the zwitterion predominates, but coexists

in dynamic equilibrium with small amounts of net negative and net positive ions. At

the exact midpoint between the two pKa values, the trace amount of net negative and

trace of net positive ions exactly balance, so that average net charge of all forms

present is zero. This pH is known as the isoelectric point pI, so pI = ½(pKa1 + pKa2).

The individual amino acids all have slightly different pKa values, so have different

isoelectric points. For amino acids with charged side-chains, the pKa of the side-

chain is involved. Thus for Asp, Glu with negative side-chains, pI = ½(pKa1 +

pKaR), where pKaR is the side-chain pKa. Cysteine also has potentially negative side-

chain with pKaR = 8.14, so pI should be calculated as for Asp and Glu, even though

the side-chain is not significantly charged at neutral pH. For His, Lys, and Arg with

positive side-chains, pI = ½(pKaR + pKa2). Amino acids have zero mobility in

electrophoresis at their isoelectric point, although this behaviour is more usually

exploited for peptides and proteins than single amino acids. Zwitterions have

minimum solubility at their isolectric point and some amino acids (in particular, with

non-polar side-chains) can be isolated by precipitation from water by adjusting the

pH to the required isoelectric point. (*) Essential only in certain cases.

87

C. MICROMOLECULES OF FATTY ACIDS

Although fatty acids occur in very large amounts as

building block components of the saponifiable lipids, only

traces occur in free (unesterified) form in cells and tissues.

Well over 100 different kinds of fatty acids have been

isolated from various lipids of animals, plants, and

microorganisms. All possess a long hydrocarbon chain and

a terminal carboxyl group. The hydrocarbon chain may be

saturated, as a in palmitic acid, or it may have one or more

double bonds, as in oleic acid; a few fatty acids contain

triple bonds. Fatty acids differ from each other primarily in

chain length and in the number and position of their

saturated bonds. They are often symbolized by a shorthand

notation that designates the length of the carbon chain and

the numbers, posistion, and configuration of the double

bonds. Thus palmitic acid (16 carbons, saturated) is

symbolized 16:0 and oleic acid [18 carbons and one double bond (cis) at carbons 9

and 10] is symbolized 18:19. it is understood that the double bonds are cis unless

indicated otherwise.

Some generalizations can be made on the different fatty acids of higher plants and

animals.

1. The most abundant have an even number of carbon atoms with chains between

14 and 22 carbon atoms long, but those with 16 or 18 carbons predominate.

88

The most common among the saturated fatty acids are palmitic acid (C16) and

stearic acid (C18).

2. Unsaturated fatty acids predominate over the saturated ones, particularly in

higher plants and in animals living at low temperatures.

3. Unsaturated fatty acids have lower melting points than saturated fatty acids of

the same chain length

4. In most monounsaturated (monoenoic) fatty acids of higher organisms there is

a double bond between carbon atoms 9 and 10. In most polyunsaturated

(polyenoic) fatty acids one double bond is between carbon atoms9 and 10; the

additional double bonds usually occur between the 9, 10 double bond and the

methy-terminal end of the chain.

5. In most types of polyunsaturated fatty acids the double bonds are seperated by

one methylene group, for example, -CH=CH-CH2-CH=CH-; only in a few

types of plant fatty acids are the double bonds in conjugation, that is, -

CH=CH-CH=CH-.

ESSENTIAL FATTY ACID

When weanling or immature rats are placed on a fat-free diet, they grow poorly,

develop a scaly skin, lose hair, and ultimately die with many pathological signs.

When linoleic acid is present in the diet, these conditions do not develop. Linolenic

acid and arachidonic acid also prevent these symptoms. Saturated and

monounsaturated fatty acids are inactive. It has been concluded that mammals can

synthesize saturated and monounsaturated fatty acids from other precursors but are

unable to make linoleic and -linoleic acids. Fatty acids required in the diet of

mammals are called essential fatty acids. The most abundant essential fatty acid in

89

mammals is lionoleic acid, which makes up from 10 to 20 percent of the total fatty

acids of their triacylglycerols and -linolenic acids

cannot be synthesized by mammals but must be obtained from plant sources, in

which they are very abundant. Linoleic acid is a necessary precursor in mammals for

the biosynthesis of arachidonic acid, which is not found in plants.

Although the specific functions of essential fatty acids in mammals were a mystery

for many years, one function has been discovered. Essential fatty acids are necessary

precursors in the biosynthesis of a group of fatty acid derivatives called

prostaglandins, homonelike compounds which in trace amounts have profound

effects on a number of important physiological activities.

STRUCTURE AND FUNCTION OF TRIACYLGLYCEROLS

(TRIGLYCERIDES)

Fatty acid esters of the alcohol

glycerol are called

acylglycerols or glycerides;

they are sometimes referred to

as "neutral fats," a term that has

become archaic. When all three

hydroxyl groups of glycerol are

esterified with fatty acids, the

structure is called a triacylglycerol. Triacyglycerols are the most abundant

family of lipids and the major components of depot or storage lipids in plant

and animal cells. Triacyglycerols that are solid at room temperature are often

referred to as "fats" and those which are liquid as "oils" Diacylglycerols (also

90

called diglycerides) and monoacylglycerols (or

monoglycerides) are also found in nature, but in much

samller amounts.Triacylglycerols occur in many

different types, according to the identity and position

of the three fatty acid components esterified to

glycerol.

1. Those with a single kind of fatty acid in all three

positions, called simple triacylglycerols, are named

after the fatty acids they contain. Examples are

tristearoylglycerol, tripalmitoylglycerol, and

trialeoyglycerol; the trivial and more commonly used

names are tristearin, tripalmitin, and triolein,

respectively.

2. Mixed triacyglycerols contain two or more different

fatty acids.

91

2.6 PHOSPHOGLYCERIDES (GLYCEROPHOSPHOLIPIDS)

The second large class of complex lipids consists of the phosphoglycerides, also

called glycerol phosphatides. They are characteristic major components of cell

membranes; only very small amounts of phosphoglycerides occur elsewhere in cells.

Phosphoglycerides are also loosely referred to as phospholipids or phosphatides, but

it should be noted that not all phosphorus-containing

lipids are phosphoglycerides; e.g., sphingomyelin is a

phospholipid because it contains phosphorus, but it is

better classified as a sphingolipid because of the nature

of the backbone structure to which the fatty acid is

attached.

In phosphoglycerides one of the primary hydroxyl

groups of glycerol is esterifed to phosphoric acid; the

other hydroxyl groups are esterfied to fatty acids. The

parent compound of the series is thus the phosphoric

ester of glycerol. This compound has an asymmetric

carbon atom and can be designated as either D-glycerol

1-phosphate or L-glycerol 3-phosphate.

Because phosphoglycerides possess a polar head

in addition to their nonpolar hydrocarbon tails, they are

called amphiphatic or polar lipids.The different types of

phosphoglycerides differ in the size, shape, and electric

charge of their polar head groups. Each type of

phosphoglyceride can exist in many different chemical species differing in their fatty

92

acid substituents. Usually there is one saturated and one unsaturated fatty acid, the

latter in the 2 position of glycerol.

The parent compound of the phosphoglycerides is phosphatidic acid, which contains

no polar alcohol head group. It occurs is only very small amounts in cells, but it is an

importnat intermediate in the biosynthesis of the phosphoglycerides. The most

abundant phosphoglycerides in higher plants and animals are

phosphatidylethanolamine and phosphatidylcholine, which contain as head groups

the amino alcohols ethanolamine and choline, respectively.

Plasmologens differ from all the other phosphoglycerides.

D MICROMOLECULES OF NUCLEIC ACIDS

PURINE AND PYRIMIDINE BASES OF NUCLEIC ACIDS.

Chemical composition

The Chemical analysis has indicated that DNA is composed of three different types

of compounds:

1. Sugar Molecule represented by a pentose sugar, the deoxyribose or 2‘-

deoxyribose.

2. Phosphoric Acid.

3. Nitrogenous Bases: These are nitrogen containing organic ring compounds.

These are of the following four types:

I. Adenine represented by – A

II. Thymine represented by – T

III. Cytosine represented by – C

93

IV. Guanine represented by – G

V.

These four nitrogenous bases are separated into two categories:

(a) Purines: These are two-ringed nitrogen compounds. Adenine and guanine are

the two purines found in DNA. Their structural formulae are represented in

fig.2.

(b) Pyrimidines: These are formed of one ring only and include cytosine and

thymine. Chemical analysis of DNA further reveals three fundamental

features described by Chargaff and is called Chargaff’s base ratio.

Molecular Structure

The constituents of DNA were isolated quite early but how these are arranged so as

to carry out their cytological and genetical activities was not known for long. DNA is

a long chain polymer was clearly understood in late 1930s. However, in 1953, D.S.

Watson and F.H.C. Crick presented a working model of DNA. This model illustrates

not only its chemical structure but also the mechanism by which it duplicates itself.

1.Nucleosides

A nitrogenous base with a molecule of deoxyribose (without phosphate group) is

known as nucleoside. The nitrogenous base is attached to first carbon atom C-1 of

deoxyribose N-glycosidic bond. In all, there are four nucleosides in a DNA

molecule. These are:

1. Adenosine – Adenine + Deoxyribose

2. Guanosine – Guanine + Deoxyribose

3. Cytidine – Cytosine + Deoxyribose

94

4. Thymidine – Thymine + Deoxyribose

2. Nucleotides ( The Monomers of DNA)

A nucleotide is formed of one molecule of deoxyribose, one molecule of phosphoric

acid and one of the four nitrogenous bases. Since there are four nitrogenous bases,

there are four type of nucleotides namely:

1. Deoxyadenylic acid -Adenine + Deoxiribose + Phosphoric acid

2. Deoxyguanylic acid -Guanine + Deoxiribose + Phosphoric acid

3. Deoxycytidylic acid -Cytosine + Deoxiribose + Phosphoric acid

4. Deoxythymidylic acid -Thymine + Deoxiribose + Phosphoric acid

1.Polynucleotide Chain (Linking of Nucleotides in a DNA Molecule)

DNA is a macromolecule formed by the linking of several thousand nucleotides.

These are called monomers or building blocks of DNA. In a nucleotide the

phosphate (phosphoric acid) molecule is attached to fifth carbon atom (C-5) of the

deoxyribose molecule through a phosphodiester linkage. The adjacent nucleotides

are connected together forming the sugar phosphate chain in which sugar and

phosphate molecule are arranged in alternate fashion. The phosphate molecule of a

nucleotide is joined to the third carbon atom of the deoxyribose. These are directed at

right angles to the long axis of the polynucleotide chain and are stacked one above

the other.

Marked Ends of Polynculeotide Chain: Each polynucleotide chain has

marked ends. Its top end has a sugar residue with free 5‘ carbon atom which is

not linked to another nucleotide. The triphosphate group is still attached to it.

95

This end is called the 5’ or 5’-P terminus. The other end of the chain ends in a

sugar residue with C-3 carbon atom not linked. It bears 3‘-OH group. This end

of polypeptide chain is called 3’ end or 3’-OH terminus . It means the

pplypeptide chains have direction and are marked as 3‘-5‘.

1.5 DOUBLE HELICAL MODEL OF DNA (WATSON & CRICK’S MODEL)

In 1953, James Watson and Francis Crick deduced the three dimensional structure

of DNA and immediately inferred its mechanism of replication, Watson and Crick

analyzed X-ray diffraction photographs of DNA fibres taken by Rosalind Franklin

and Maurice Klilkins and derived a structural model that has proved to be essentially

correct.

The salient features of their model are:-

1. Two helical polynucleotide chains are coiled around common axis, the chains

run in the opposite directions.

2. The purine and pyrimidine bases are on the inside of the helix, where as the

phosphate and deoxyribose units are on the outside the planes of the bases are

perpendicular to the helix axis. The planes of the sugars are nearly at right

angles to those of the bases.

3. The diameter of the helix is 20 A0, adjacent bases are separated by 34 A

0 along

the helix and related by a rotation of 360, hence the helical structure repeats

after in residues on each chain, i.e., at interval of 34A0.

4. The two chains are held together by hydrogen bonds between the pairs of

bases adenine is always paired with thymine guanine is always paired width

cytosine.

5. The sequence of bases along a polynucleotide chain is not restricted in any

way, the precise sequence of bases carries the genetic information.

6. The ratio of A+G/C+T always equals to one

96

7. In every organism, the sequence of nucleotides in constant. The ratio of

A=T/G=C is also specific to organisms.

8. Each pitch of DNA has two major and two minor groves.

Fig. DNA Structure

The most important, aspect of the DNA double helix is the specificity of the pairing

of the bases. Watson and Crick deduced that adenine must pair with thymine and

guanine with cytosine.

97

CHECK YOUR PROGRESS 2

1.10 LET US SUM UP

The cell is the basic unit of life. There are millions of different types of cells.

There are cells that are organisms onto themselves, such as microscopic amoeba and

bacteria cells. And there are cells that only function when part of a larger organism,

such as the cells that make up your body. The cell is the smallest unit of life in our

bodies. In the body, there are brain cells, skin cells, liver cells, stomach cells, and

the list goes on. All of these cells have unique functions and features. And all have

some recognizable similarities. All cells have an outer covering called the plasma

Note: Write your answer in the space given below.

Check your answer with the one at the end of the unit.

Fill in the blanks.

1 Lipids are ________ of higher fatty acids.

2 Fluid mosaic model of membrane was proposed by ________

3 DNA and RNA are -------------------.

4 Adinine and guanine are------------------.

5 Term ATP stands for --------------------.

Write short notes on

A) glycolysis .b) building blocks of biomolecules

-------------------------------------------------------------------------------------------

-------------------------------------------------------------------------------------------

---------------------------------------------------------------------------------------------

-----------------------------------------------------------------------------------------------

98

membrane, protecting it from the outside environment. The cell membrane regulates

the movement of water, nutrients and wastes into and out of the cell. Inside of the

cell membrane are the working parts of the cell. At the center of the cell is the cell

nucleus. The cell nucleus contains the cell's DNA, the genetic code that coordinates

protein synthesis. In addition to the nucleus, there are many organelles inside of the

cell - small structures that help carry out the day-to-day operations of the cell. One

important cellular organelle is the ribosome. Ribosomes participate in protein

synthesis. The transcription phase of protein synthesis takes places in the cell

nucleus. After this step is complete, the mRNA leaves the nucleus and travels to the

cell's ribosomes, where translation occurs. Another important cellular organelle is

the mitochondrion. Mitochondria (many mitochondrion) are often referred to as the

power plants of the cell because many of the reactions that produce energy take place

in mitochondria. Also important in the life of a cell are the lysosomes. Lysosomes

are organelles that contain enzymes that aid in the digestion of nutrient molecules

and other materials

There are many different types of cells. One major difference in cells occurs

between plant cells and animal cells. While both plant and animal cells contain the

structures discussed above, plant cells have some additional specialized structures.

Many animals have skeletons to give their body structure and support. Plants have a

unique cellular structure called the cell wall. The cell wall is a rigid structure outside

of the cell membrane composed mainly of the polysaccharide cellulose. The cell wall

gives the plant cell a defined shape which helps support individual parts of plants. In

addition to the cell wall, plant cells contain an organelle called the chloroplast. The

chloroplast allow plants to harvest energy from sunlight. Specialized pigments in the

chloroplast (including the common green pigment chlorophyll) absorb sunlight and

use this energy to complete the chemical reaction

99

Eukaryotic cells are about 15 times wider than a typical prokaryote and can be as

much as 1000 times greater in volume. The major difference between prokaryotes

and eukaryotes is that eukaryotic cells contain membrane-bound compartments in

which specific metabolic activities take place. Most important among these is a cell

nucleus, a membrane-delineated compartment that houses the eukaryotic cell's DNA.

This nucleus gives the eukaryote its name, which means "true nucleus." Other

differences include:

The plasma membrane resembles that of prokaryotes in function, with minor

differences in the setup. Cell walls may or may not be present.

The eukaryotic DNA is organized in one or more linear molecules, called

chromosomes, which are associated with histone proteins. All chromosomal

DNA is stored in the cell nucleus, separated from the cytoplasm by a

membrane. Some eukaryotic organelles such as mitochondria also contain

some DNA.

Many eukaryotic cells are ciliated with primary cilia. Primary cilia play

important roles in chemosensation, mechanosensation, and thermosensation.

Cilia may thus be "viewed as sensory cellular antennae that coordinate a large

number of cellular signaling pathways, sometimes coupling the signaling to

ciliary motility or alternatively to cell division and differentiation."[7]

Eukaryotes can move using motile cilia or flagella. The flagella are more

complex than those of prokaryotes.

100

INTRACELLULAR ORGANELLS AND THEIR FUNCTIONS

Golgi Apparatus

An Italian neurologist (i.e., physician) Camillo Golgi in 1873 discovered, them

which is commonly known as the Golgi bodies. The Golgi apparatus occurs in all

cells except the prokaryotic cells (viz., mycoplasmas, bacteria and blue green algae)

and eukaryotic cells of certain fungi, sperm cells of bryophytes and pteridiophytes,

cells of mature sieve tubes of plants and mature sperm and red blood cells of

animals. In animal cells, there usually occurs a single Golgi apparatus, however,

its number may vary from animal to animal and from cell to cell. Thus Paramoeba

species has two Golgi apparatus and nerve cells, liver cells and chordate oocytes

have multiple Golgi apparatuses, there being about 50 of them in the liver cells. The

Golgi apparatus is morphologically very similar in both plant and animal cells.

However, it is extremely pleomorphic: in some cell types it appears compact and

limited, in others spread out and reticular (net-like). Its shape and form may very

depending on cell type.

Lysosomes

The lysosomes (Gr. lyso=digestive + soma = bodies) are tiny membrane-bound

vesicles involved in intracellular digestion. They contain a variety of hydrolytic

enzymes that remain active under acidic conditions.

The lysosomes occur in most animal and few plant cells. They are

absent in bacteria and mature mammalian ertythrocytes. Few lysosomes occur in

muscle cells or in cells of the pancreas. Leucocytes, especially granulocytes are a

particularly rich source of lysosomes. Their lysosomes are so large sized that they

101

can be observed under the light microscope. Lysosomes are also numerous in

epithelial cells of absorptive, secretory and excretory organs (e.g., intestine, liver,

kidney, etc.) They occur in abundance in the epithelial cells of lungs and uterus.

Lastly phagocytic cells and cells of reticuloendothelial system (e.g., bone marrow,

spleen and liver) are also rich in lysosomes.The lysosomes are round vacuolar

structure which remain filled with dense material and are bounded by single unit

membrane. , a lysosome may contain up to 40 types of hydrolytic enzymes . The so-

called latency of the lysosomal enzymes is due to the presence of the membrane

which is resistant to the enzymes that it encloses. Lysosomes are extremely dynamic

organelles,

Endoplasmic Reticulum

The cytoplasmic matrix is traversed by a complex network of inter-connecting

membrane bound vacuoles or cavities. often remain concentrated in the endoplasmic

portion of the cytoplasm ; therefore, known as endoplasmic reticulum, a name

derived from the fact that in the light microscope it looks like a "net in the cytoplasm

Morphologically, the endoplasmic reticulum may occur in the following three

forms : 1. Lamellar form or cisternae (A closed, fluid-filled sac, vesicle or cavity is

called cisternae) 2. vesicular form or vesicle and 3. tubular form or tubules.

The cavities of cisternae, vesicles and tubules of the endoplasmic reticulum

are bounded by a thin membrane of 50 to 60 A0 thickness. The membrane of

endoplasmic reticulum is fluid-mosaic like the unit membrane of the plasma

membrane, nucleus, Golgi apparatus, etc. The membrane, thus, is composed of a

bimolecular layer of phospholipids in which 'float' proteins of various sorts.

102

Two types of endoplasmic reticulum have been observed in same or different

types of cells which are as follows :1.Agranular or Smooth Endoplasmic

Reticulum and Granular of Rough Endoplasmic Reticulum

The membranes of the endoplasmic reticulum are found to contain

many kinds of enzymes which are needed for various important synthetic activities,

steroids and hexose metabolism. The enzymes of the endoplasmic reticulum perform

the following important functions :

The endoplasmic reticulum acts as secretory, storage, circulatory and nervous

system for the cell. performs following important functions :

Microbodies : Structure and Types

Microbodies are spherical or oblate in form. They are bounded by a single

membrane and have an interior or matrix which is amorphous or granular Micrbodies

are most easily distinguished from other cell organelles by their content of catalase

enzyme. Catalase can be visualized with the electron microscope when cells are

treated with the stain DAB (i.e., 3,3'-diaminobenzidine). The product is electron

opaque and appears as dark regions in the cell where catalase is present

Peroxisomes

Peroxisomes occur in many animal cells and in a wide range of plants. They

are present in all photosynthetic cells of higher plants in etiolated leaf tissue, in

coleoptiles and hypocotyls, in tobacco stem and callus, in ripening pear fruits and

also in Euglenophyta, Protozoa, brown algae, fungi liverworts, mosses and ferns.

103

Peroxisomes are variable in size and shape, but usually appear circular in cross

section having diameter between 0.2 and 1.5m (0.2 and 0.25 m diameter in most

mammalia

Glyoxysomes.

Glyoxysomes are found to occur in the cells of yeast, Neurospora, and oil rich

seeds of many higher plants. They resemble with peroxisomes in morphological

details, except that, their crystalloid core consists of dense rods of 6.0 m diameter.

They have enzymes for fatty acid metabolism and gluconeogenesis, i.e. conversion

of stored lipid molecules of spherosomes of germinating seeds into the molecules of

carbohydrates.

MITOCHONDRIA

In cell biology, a mitochondrion (plural mitochondria) is a membrane-enclosed

organelle found in most eukaryotic cells. These organelles range from 0.5 to

10 micrometers (μm) in diameter. Mitochondria are sometimes described as "cellular

power plants" because they generate most of the cell's supply of adenosine

triphosphate (ATP), used as a source of chemical energy.

The most prominent roles of mitochondria are to produce ATP (i.e., phosphorylation

of ADP) through respiration, and to regulate cellular metabolism.[7]

The central set of

reactions involved in ATP production are collectively known as the citric acid cycle,

or the Krebs Cycle. However, the mitochondrion has many other functions in

addition to the production of ATP.

.

104

COMPARISION OF PLANT AND ANIMAL CELLS

Comparision of plant and animal cells.

Animal Cell Plant Cell

Cilia: Present It is very rare

Shape: Round (irregular

shape)

Rectangular (fixed

shape)

Chloroplast: Animal cells don't have

chloroplasts

Plant cells have

chloroplasts because

they make their own

food

Vacuole:

One or more small

vacuoles (much

smaller than plant

cells).

One, large central

vacuole taking up 90%

of cell volume.

Centrioles: Present in all animal

cells

Only present in lower

plant forms.

Plastids: Absent Present

Cell wall: Absent Present

Plasma Membrane: only cell membrane cell wall and a cell

membrane

Lysosomes: Lysosomes occur in

cytoplasm.

Lysosomes usually not

evident.

105

During synthesis of metabolites energy is required and such constructive

reactions are called anabolism.Example photosynthesis

Co2 +h2o+Ribulose 1,5, diphospate- Rudp carbooxylase--------- 2 mol of 3 PGA

Where as reactions in which break down of certain metabolites occurs and energy

is released are called catabolism.Example Glycolysis,krebs cycle..

Origin of the first cell

There are several theories about the origin of small molecules that could lead to life

in an early Earth. One is that they came from meteorites Another is that they were

created at deep-sea vents. A third is that they were synthesized by lightning in a

reducing atmosphere); although it is not clear if Earth had such an atmosphere. There

are essentially no experimental data defining what the first self-replicating forms

were. RNA is generally assumed to be the earliest self-replicating molecule, as it is

capable of both storing genetic information and catalyzing chemical reactions (see

RNA world hypothesis). But some other entity with the potential to self-replicate

could have preceded RNA, like clay or peptide nucleic acid.

The living organisms are composed of molecules which are intrinsically

inanimate.These molecules confer a remarkable combination of characteristics called

life.Many of he most important molecules in biological systems are polymers,that is

large molecules made up of smaller subunits joined together by covalent bonds,and

in some cases in a specific order. Most of the molecular constituents of living

106

systems are composed of C-atoms joined with other carbon atoms and with hydrogen

,oxygen or nitrogen.Thus form a great variety of molecules such as aminoacids

,monosaccharides,nucleotides and fattyacids.These are called micromolecules.These

micromolecules serve as monomeric subunits of proteins,nucleic acids,

polysaccharides and lipids respectively which are designated as

macromolecules.These macromolecules serve more than one functions in living

cells and in all living organisms.

2.1 CHECK YOUR PROGRESS ;THE KEY

KEY 1

1 single

2 Bacteria E. coli

3Membrane bound organelles and nucleus

4 power house of the cell.

5 suicidal sacs

6 cellulose

7 glycogen

8 absent

KEY2

1 esters

2Sanger and Nicholsan

3Nucleic acid

4 Purines

5Adinine tri phosphate

107

2.2 ASSIGNMENT / ACTIVITY

1 Explain in detail the structure of prokaryotic cell.

2 Compare the structure of prokaryotic and eukaryotic cells.

3 Describe the structure of plant and animal cell.

4 Write short notes on cell organelles,origin of life, biomolecules, ATP .

2.3 REFERENCES

1 BIOCHEMISTRY BY LEHININGER

2 BIOCHEMISTRY BY STRYER

3 CELL BIOLOGY BY RASTOGI

4 CELL ANDMOLECULAR BIOLOGY BY AJOY PAUL

5 CELL BIOLOGY BY S. CHANDRA ROY

6 MOLECULAR BIOLOGY BY ROBERTIES DE ROBERTIES.

108

UNIVERSITY - MADHYA PRADESH BHOJ OPEN

UNIVERSITY BHOPAL (M.P.)

PROGRAMME - M.Sc.Chemistry (Previous)

PAPER - V (A-II)

TITLE OF PAPER - BIOLOGY FOR CHEMIST

BKOCK NO . - II

UNIT WRITER - UNIT - I Smt. Shikha Mandloi

Asst. Prof. Microbiology

Sri Sathya Sai College for Women

UNIT – II Smt. Shikha Mandloi

Asst. Prof. Microbiology

Sri Sathya Sai College for Women

EDITOR - Dr.(Smt.)Renu Mishra,HOD,Botany &

Microbiology,Sri Sathya Sai College for

Women, Bhopal

COORDINATION

COMMITTEE - Dr. Abha Swarup, Director, Printing & Translation

Major Pradeep Khare, Consultant, Printing &

Translation

POST GRADUATE PROGRAMME

M.Sc. CHEMISTRY( PREVIOUS)

DISTANCE EDUCATION SELF INSTRUCTIONAL MATERIAL

109

Paper-V(A-II)

BIOLOGY FOR CHEMISTS

BLOCK :II

MADHYA PRADESH BHOJ OPEN UNIVERSITY

BHOPAL (M.P.)

UNIT II

Carbohydrates

Introduction

The carbohydrates, or saccharides, are most simply defined as polyhydroxy aldehydes or ketones

and their derivatives. Monosaccharides, consist of a single polyhydroxy aldehyde or ketone unit.

The most abundant monosaccharide is the six-carbon D-glucose; it is the parent monosaccharide

from which most others are derived. D-Glucose is the major fuel for most organisms and the basic

building block of the most abundant polysaccharides, such as starch and cellulose.Oligosaccharides

(Greek oligo, ―few‖) contain from two to ten monosaccharide units joined in glycosidic linkage.

Polysaccharides contain many monosaccharide units joined in long linear or branched chains. Most

polysaccharides contain recurring monosaccharide units of only a single kind or two alternating

kinds.Polysaccharides have two major biological functions, as a storage form of fuel and as

structural elements. In the biosphere there is probably more carbohydrate than all other organic

matter combined. Starch is the chief form of fuel storage in most plants, whereas cellulose is the

main extracellular structural component of the rigid cell walls and the fibrous and woody tissues of

plants. Glycogen, which resembles starch in structure, is the chief storage carbohydrate in animals.

Others polysaccharides serve as major components of the cell walls of bacteria and of the soft cell

coats in animal tissues.

Lipids are esters of higher fatty acids. Lipids are water-insoluble organic biomolecules that

can be extracted from cells and tissues by nonpolar solvents, e.g., choloroform, ether, or benzene.

110

There are several different families or classes of lipids but all derive their distinctive properties

from the hydrocarbon nature of a major portion of their structure. (1) as structural components of

membranes, (2) as storage and transport forms of metabolic fuel, (3) as a protective coating on the

surface of many organisms, and (4) as cell-surface components concerned in cell recognition,

species specificity, and tissue immunity. Some substances classified among the lipids have intense

biological activity; they include some of the vitamins and hormones.Although lipids are a distinct

class of biomolecules, we shall see that they often occur combined, either covalently or through

weak bonds, with members of other classes of bio-molecules to yield hybrid molecules such as

glycolipids, lipoproteins, which contain both lipids and proteins. In such biomolecules the

distinctive chemical and physical properties of their components are blended to fill specialized

biological functions.

CARBOHYDRATES

1.0 Introduction

1.1 Objectives

1.2 Configuration of Monosaccharides and their family

1.3 Structure and function of important derivatives of monosaccharides

1.3.1 Glycosides

1.3.2 Deoxysugars

1.3.3 Myoinositol

1.3.4 Aminosugars

1.3.5 N-acetyle muramic acid

1.3.6 Sialic acid

1.3.7 Disaccharides

1.3.8 Polysaccharide

1.4 Structural Polysaccharides

1.4.1 Cellulose and ehitin

1.5 Storage polysaccharides

1.5.1 Starch and Glycogen

1.6 Structure and biological functions of glucosaminoglycans or

mucopolysaccharides

1.7 Carbohydrates of glycoprotiens and glycolipids

111

1.8 Role of sugars in biological recognition blood group substance – Ascorbic

acid

1.9 Carbohydrates metabolism

1.9.1 Kreb’s cycle

1.9.2 Glycolysis

1.9.3 Glycogenesis

1.9.4 Glycogenolysis

1.9.5 Gluconeogenesis

1.9.6 Pentose Phosphate pathway.

2.0 Let us sum up

2.1 Check your progress key

2.2 Assignment/Activity

2.3 References

CARBOHYDRATE

1.0 INTRODUCTION

The carbohydrates, or saccharides, are most simply

defined as polyhydroxy aldehydes or ketones and their

derivatives. Many have the empirical formula (CH2O)n,

which originally suggested they were ―hydrates‖ of

carbon. Monosaccharides, also called simple sugars,

consist of a single polyhydroxy aldehyde or ketone unit.

The most abundant monosaccharide is the six-carbon

D-glucose; it is the parent monosaccharide from which

most others are derived. D-Glucose is the major fuel for

most organisms and the basic building block of the

112

most abundant polysaccharides, such as starch and cellulose.

Oligosaccharides (Greek oligo, ―few‖) contain from two to ten monosaccharide units

joined in glycosidic linkage. Polysaccharides contain many monosaccharide units

joined in long linear or branched chains. Most polysaccharides contain recurring

monosaccharide units of only a single kind or two alternating kinds.

Polysaccharides have two major biological functions, as a storage form of fuel and as

structural elements. In the biosphere there is probably more carbohydrate than all

other organic matter combined, thanks largely to the abundance in the plant world of

two polymers of D-glucose, starch and cellulose. Starch is the chief form of fuel

storage in most plants, whereas cellulose is the main extracellular structural

component of the rigid cell walls and the fibrous and woody tissues of plants.

Glycogen, which resembles starch in structure, is the chief storage carbohydrate in

animals. Others polysaccharides serve as major components of the cell walls of

bacteria and of the soft cell

coats in animal tissues.

1.1 OBJECTIVES

FAMILY OF

MONOSACCHARIDES

Monosaccharides have the

empirical formula (CH2O)n,

where n=3 or some larger

number. The carbon skeleton

of the common

monosaccharides is

unbranched and each carbon

Fig. 1

113

atom except one contains a hydroxyl group; at the remaining carbon there is a

carbonyl oxygen, which, as we shall see, is often combined in an acetal or ketal

linkage. If the carbonyl groups is at the end of the chain, the monosaccharide is an

aldehyde derivative and called an aldose; if it is at any other position, the

monosaccharides is a ketone derivative and called ketose. The simplest

monosaccharides are the three carbon trioses glyceraldehydes and dihydrosyacetone.

Glyceraldehyde is an aldotriose; dihydroxyacetone is a ketotriose. Also among the

monosaccharides are the tetroses (four carbons), pentoses (five carbons), hexoses

(six carbons), heptoses (seven carbons), and octoses (eight carbons). Each exists in

two series, i.e., aldotetroses and ketotetroses, aldopentoses and ketopentoses,

aldohexoses and ketohexoses, etc. The structures of D aldoses and D ketoses are

shown. In both classes of monosaccharides the hexoses are by far the most abundant.

However, aldopentoses are important components of nucleic acids and various

polysaccharides; derivatives of trioses and heptoses are important intermediates in

carbohydrate metabolism. All the simple monosaccharides are white crystalline

solids that are free soluble in water but insoluble in nonpolar solvents. Most have

sweet taste.

1.2 CONFIGURATION OF MONOSACCHARIDES & THEIR FAIMILY

First, we must clarify two terms often confused. Configuration denotes the

arrangement in space of substituent groups in stereoisomers such structures cannot

be inter-converted without breaking one or more covalent bonds. Conformation

refers to the spatial arrangement of substituent groups that are free to assume many

different positions, without breaking bonds, because of rotation about the single

bonds in the molecule. In the hydrocarbon ethane, for example, one might expect

complete freedom of rotation around the C-C single bond to yield an infinite number

114

of conformations of the molecule. However, the staggered conformation is more

stable than all others and thus predominates, whereas the eclipsed form is least

stable.

All the monosaccharides expect

dihydroxyacetone contain one or

more asymmetric carbon atoms and

thus are chiral molecules.

Glyceraldehyde contains only one

asymmetric carbon atom and

therefore can exist as two different

stereoisomers (Figure- 4). It will be

recalled that C- and L-

Glyceraldehyde are the reference, or

parent, compounds for designating

the absolute configuration of all

stereoisomeric compounds.

Aldotetroses have two asymmetric

carbon atoms and aldopentoses have

three. The aldohexoses have four

asymmetric carbon atoms and thus

exist in the form of 2n = 2

4 = 16

different stereoisomers, 8 of which

are shown in Figure- 2. As expected, the monosaccharides with asymmetric carbon

atoms are optically active. For example, the usual form of glucose found in nature in

dextrorotatory ([α]20

= +52.70), and the usual form of fructose is levorotatory ([α]

20 =

-92.40), but both are members of the D series since their absolute configurations are

related to D-glyceraldehyde. For sugars having two or more asymmetric carbon

Fig. 3

D

115

Fig-4 The stereoisomers of

glyceraldehyde, showing projection

formulas (top) and perspective

formulas (bottom).

atoms, the convention has been adopted that the prefixes D and L refer to the

asymmetric carbon atoms farthest removed from carbonyl carbon atom.

Fig4 shows the projection formulas of the D aldoses. All have the same

configuration at the asymmetric carbon atom farthest from the carbonyl carbon, but

because most have two or more asymmetric

carbon atoms, a number of isomeric D aldoses

exist, most important biologically being D-

glyceraldehyde. D-ribose, D-glucose, D-

mannose, and D-galactose.

Figure-4 shows the projection formulas of the

D ketoses; all share the same configuration at

the asymmetric carbon atom farthest from the

carbonyl group. Ketoses are sometimes

designated by inserting ul into the name of the

corresponding aldose; e.g., D-ribulose is the

ketopentose corresponding to the aldopentose

D-ribose. The most important ketoses biologically and dihydroxyacetone. D-

ribulose, and D-fructose.

Aldoses and ketoses of the L series are mirror images of their D counterparts. L

sugars are found in nature, but they are not so abundant as D sugars. Among the

most important are L-fucose, L-rhamnose , and L-sorbose.

Two sugars differing only in the configuration around one specific carbon atom are

called epimers of each other. Thus, D-glucose and D-mannose are epimers with

116

respect to carbon atom 2, and D-glucose and D-galactose are epimers with respect to

carbon atom 4.

1.3 STRUCTURE AND FUNCTION OF IMPORTANT DERIVATIVES OF

MONOSACCHARIDES

1.3.1 GLYCOSIDES

Aldopyronoses readily react with alcohols in the presence of a mineral acid to form

anomeric α- and β-glycosides. The glycosides are asymmetric mixed acetals formed

by the reaction of the anomeric carbon atoms of the intramolecular hemiacetal or

pyranose form of the aldohexose with a hydroxyl group furnished by an alcohol.

This is called a glycosidic bond. The aromeric carbon in such glycosides is

asymmetric. D- -D-glycopyranoside ([α] =

158.90), and methyl β -D-glucopyranoside ([α] = -34.2

0).

The glycosidic linkage is also formed by the reaction of the numeric carbon of a

monosaccharide with a hydrosyl group of another monosaccharide to yield a

disaccharide. Oligosaccharides and polysaccharides are chains of monosaccharides

joined by glycosidic linkages. The glycosidic linkage is stable to bases but is

hydrolyzed by boiling with acid to yield the free monosaccharide and free alcohol.

Glycosides are also hydrolyzed by enzymes called glycosidases, which differ in their

specificity according to the type of glycosidic bond (α or β), the structure of the

monosaccharide unit (s), and the structure of the alcohol.

Whether a given glycoside exists in furanose or pyranose form can be ascertained by

osicative degradation with periodic acid, which cleaves 1,2-dihydroxy compounds.

Treatment of methyl α -D-gludopyranoside with periodate cleaves the pyranose ring

117

Fig- 5

to yield a dialehyde and formic acid. (Figure 10-14). Periodate cleavage of methyl α

-D-arabinofuranoside yields the same dialdehyde but no formic acid.

1.3.2 DEOXYSUGARS

Several deoxy sugars are found in

nature. The most abundant is 2-

deoxy-D-ribose, the sugar component

of deoxyribonucleic acid. L-

Rhamnose (6-deoxy-L-mannose) and

L-fructose (6-deoxy-L-galactose) are

important components of some

bacterial cell walls.

1.3.3 SUGAR ALCOHOLS - MYOINOSITOL

The carbonyl group of monosaccharides

can be reduced by H2 gas in the presence of

metal catalysts or by sodium amalgam in

water to form the corresponding sugar

alcohols, D-Glucose, for example Yields

the sugar alcohol D-glucitol, also formed

by reduction of L-sorbose and often called

L-sorbitol. D-Mannose yields D-

118

Fig- 6

Fig- 7

mannitol. Such reductions can also be carried out by enzymes.

Two other sugar alcohols occur in nature in some abundance. One is glycerol, an

important component of some lipids. The other is the fully hydroxylated cyclohexane

derivateive inositol, which can exist in

several stereoisomeric forms. One of the stereoisomers of inositol, myo-inositol, is

found not only in the lipid phosphatidylinositol but also in phytic acid, the

hexaphosphoric ester of inositol. The calcium-magnesium salt of phytic acid is called

phytin; it is abundant in the extracellular supporting material in higher-plant tissues.

The structures of some sugar alcohols are shown in fig.6

1.3.4 AMINO SUGARS

Two amino sugars of wide distribution are D-

glucosamine (25-amino-2-deoxy-D-glucose) and

D-galactosamine (2-amino-2-deoxy-D-

galactose), in which the hydroxyl groups at

carbon atom 2 is replaced by an amino group. D-

glucosamine occurs in many polysaccharides of

vertebrate tissues and is also a major component

of chitin, a structural polysaccharide found in the

exoskeletons of insects and crustaceans. D-

Galactosamine is a component of glycolipids and

of the major polysaccharide of cartilage,and

chondroitin sulfate.

1.3.5 MURAMIC ACID AND NEURAMINIC ACID AND SIALIC ACID

119

Fig- 8

These sugar derivatives are

important building blocks of the

structural polysaccharides found in

the cell walls of bacteria and the cell

coats of higher-animal cells

respectively. Both are nine-carbon

amino sugar derivatives; they may

be visualized as consisting of a six-

carbon amino

sugar linked to a three-carbon sugar

acid; the amino group is usually acetylated. N-Acetylmuramic acid is a major

building blocks of the polysaccharide back-bone of bacterial cell walls. It consists of

N-acetyl-D-glucosamine

in ether linkage with the

three-carbon- D-lactic acid. N-Acetylneuraminic acid is derived from N-acetyl-d-

mannosamine and pyruvic acid. It is an important building block of the

oligosaccharide chains found in the glycoproteins and glycolipids of the cell coasts

and membranes of animal tissues. N-Acyl derivatives of neuraminic acid are

generically called sialic acids. The sialic acids found in human tissues contain an N-

acetyl group; in some other species they contain an N-glycolyl group.

1.3.6 DISACCHARIDES

Disaccharides consist of two monosaccharides joined by a glycosidic linkage. The

most common disaccharides are maltose, lactose, and sucrose. Maltose, which is

formed as an intermediate product of the action of amylases on starch, contains two

D-glucose residues. It is a mixed acetal of the anomeric carbon atom 1 of D-glucose;

one hydroxyl group is furnished intramolecularly by carbon atom 5 and the other by

120

Fig- 9

carbon atom 4 of a second D-glucose molecule. But glucose moieties are in pyronose

form, and the configuration at the anomeric carbon atom in glycosidic linkage is α.

Maltose may therefore may therefore be called O- α -D-glucopyranosyl-(14)-α-D-

glucopyranose. The second glucose residue of maltose had a free anomeric carbon

atom capable of existing in α β forms; both the α and β forms are products of

enzyme action. The first glucose residue cannot undergo oxidation, but the second

residue can; it is called the reducing end. The position of the glycosidic linkage

between the two glucose residues is symbolized 1 4. Exhaustive methylation

of all the free hydroxyl groups, followed by hydrolysis of the glycosidic linkage, has

proved that the glycosidic linkage in maltose involves carbon atom 1 of the first

residue and carbon atom 4 of

the second glucose unit. The

resulting methylated fragments

were 2,3,4,6-tetra-O-methyl-D-

glucose and 2,3,6-tri-O-methyl-

D-glucose.

Two other common

disaccharides that contain two

D-glucose units are cellobiose

and gentiobiose. Cellobiose, the

repeating disaccharide unit of

cellulose, has a β(14)

glycosidic linkage; its full name

is thus O-β-D-glucopyranosyl-

(14)-β-D-

glucopyranose. In

121

gentiobiose, the glycosidic linkage is β (16). Since both these disaccharides have a

free anomeric carbon, they are reducing sugars.

The disaccharide lactose [O- β -D-glucopyranosyl-(14)- β-D-glucopyranose] is

found in milk but otherwise does not occur in nature. It yields D-galactose and D-

glucose on hydrolysis. Since it has free anomeric carbon on the glucose residue,

lactose is a reducing disaccharide.

Sucrose, is a disaccharide of glucose and fructose [O- β -D-fructofuranosyl-(21)- α

-D-glucopyranoside]. It is extremely abundant in the plant world and is familiar as

table sugar. Unlike most disaccharides and oligosaccharides, sucrose contains no free

anomeric carbon atom; the anomeric carbon atoms of the two hexoses are linked to

each other. For this reason sucrose does not undergo mutarotation, does not react

with phenylhydrazine to form osazones, and does not act as a reducing sugar. It is

much more readily hydrolyzed than other disaccharides.

1.3.7 POLYSACCHARIDES (GLYCANS)

Most of the carbohydrates found in nature occur as polysaccharides of high

molecular weight. On complete hydrolysis with acid or specific enzymes, these

polysaccharides yield monosaccharides or simple monosaccharide derivatives. D-

Glucose is the most prevalent monosaccharide unit in polysaccharides, but

polysaccharides of D-mannose, D-fructose, D- and L-galactose, D-xylose, and D-

arabinose are also common. Monosaccharide derivatives commonly found as

structural units of natural polysaccharides are D-glucosamine, D-galactosamine, D-

glucuronic acid, N-acetyl-muramic acid, and N-acetylneuraminic acid.

Polysaccharides, which are also called glycans, differ in the nature of their recurring

monosaccharide units, in the length of their chains, and in the degree of branching.

122

They are divided into homopolysaccharides, which contains only one type of

monomeric unit, and heteropolysaccharides, which contain two or more different

monomeric units. Starch, which contains only D-glucose units, is a

homopolysaccyharide. Hyaluronic acid consists of alternating residues of D-

glucuronic acid and N-acetyl-D-glucosamine and is thus a heteropolysaccharide.

Homopolysaccharides are given class names indicating the nature of their building

blocks. For example, those containing D-glucose units, e.g., starch and glycogen, are

called glucans and those containing mannose units are mannans. The important

polysaccharides are best described in terms of their biological function.

1.4 STRUCTURAL POLYSACCHARIDES

Many polysaccharides serve primarily as structural elements in cell walls and coats,

intercellular spaces, and connective tissue, where they give shape, elasticity, or

rigidity to plant and animal tissues as well as protection and support to unicellular

organisms. Polysaccharides also are found as the major organic compounds of the

exoskeletons of many invertebrates. For example, the polysaccharide chitin, a

homopolymer of N-acetyl-D-glucosamine in β(14) linkage, is the major organic

element in the exoskeleton of insects and crustacean.

Cell walls and coats are not only important in maintaining the structure of tissues but

also contain specific cell-cell recognition sites important in the morphogenesis of

tissues and organs. They also contain other protective elements, such as the cell-

surface antibodies of vertebrates tissues. For this reason we shall examine the

123

structural polysaccharides in the context of the molecular organization of cell walls

and coats.

1.4.1 Plant Cell Walls- Cellulose & Chitin

Since plant cells must be able to withstand the large osmotic pressure difference

between the extracellular and intracellular fluid compartments, they require rigid cell

walls to keep from sweeling. In larger plants and trees the cell walls not only must

contribute physical strength or rigidity to stems, leaves, and root tissues but must

also be able to sustain large weights.

The most abundant cell-wall and structuaral polysaccharide in the plant world is

cellulose, a linear polymer of D-glucose in β (14) linkages. Cellulose is the major

component of wood and thus of paper; cotton is nearly pure cellulose. Cellulose is

also found in some lower invertebrates. It is almost entirely of extracellular

occurrence.

On complete hydrolysis with strong acids, cellulose yields only D-glucose, but

partial hydrolysis yield the reducing disaccharide cellobiose , in which the linkage

between the D-glucose units is β(14). When cellulose is exhaustively methylated

and then hydrolyzed, it yields only 2,3,6-tri-O-methylglucose, showing not only that

all its glycosidic linkage are (14) but also that there are no branch points. The only

chemical difference between starch and cellulose, both homopolysaccharides of D-

glucose, is that starch α (14) linkages and cellulose β (14). Cellulose is not

attacked by either α β β

(14) linkages of cellulose are not secreted in the digestive tract of most mammals,

and they cannot use cellulose for food. However, the ruminants, e.g., the cow, are an

exception: they can utilize cellulose as food since bacteria in the rumen form the

enzyme cellulase, which hydrolyzes cellulose to D-glucose.

124

The minimum molecular weight of cellulose from different sources has been

estimated to vary from about 50,000 to 2,500,000 in different species, equivalent to

300 to 15,000 glucose residues. X-ray diffraction analysis indicates that cellulose

molecules are organized in bundles of parallel chains to form fibrils. Although

cellulose has a high affinity for water, it is completely in soluble in it.

In the cell walls of plants densely packed cellulose fibrils surround the cell in regular

parallel arrays, often in criss-cross layers. These fibrils are cemented together by a

matrix of three other olymeric materials: hemicellulose, pectin, and extensin.

Hemicelluloses are not related structurally to cellulose by are polymers of pentoses,

particularly D-xylans, polymers of D-xylose in β(14) linkage with side chains of

arabinose and other sugars. Pectin is a polymer of methyl D-galacturonate. Extensin,

a complex glycoprotein, is attached covalently to the cellulose fibrils. Extensin

resumbles its animals tissue counterpart collagen in beng rich in hydroxyproline

residues; it also contains many side chains with arabinose and galactose residues.

The cell walls of higher plants can be compared to cases of reinforced concrete, in

which the cellulose fibrils correspond to the steel rods and the matrix materials to the

concrete. These walls are capable of withstanding enormous weights and physical

stress. Wood contains another polymeric substance, lignin, which makes up nearly

25 percent of its dry weight. Lignin is a polymer of aromatic alcohols.

Other polysaccharides serving as cell-wall or structural components in plants include

agar of seaweeds, which contains D- and L- galactose residues, some of which are

esterified with sulfuric acid; alginic acid of algae and kelp, which contains D-

mannuronic acid units; and gum arabic, a vegetable gum, which contains D-galactose

and D-glucuronic acid residues, as well arabinose and rhamnose.

CHITIN

Those amino sugars which occur in nature in combination with protein and

glucosamine, are called chitin. Generally, it is made up of chitibiose, a disachharide,

125

which on decomposition yield N-acytyl glucosamine when chitin is hydrolysed by

acids, it yields acetic-acid and glucosamine, it occurs in shells of arthropods, lenses

of eyes, lining of digestive tract, respiratory and excretory tract of insects.

Bacterial Cell Walls

The cell walls of bacteria are rigid, porous, boxlike structures that provide physical

protection to the cell. Since bacteria have a high internal osmotic pressure, and since

they are often exposed to a quite variable and sometimes hypotonic external

environment, they must have a rigid cell wall to prevent swelling and rupture of the

cell membrane. The structure and biosynthesis of bacterial cell walls have been

intensively studied.

The covalently linked framework of the cell wall actually may be regarded as a

single large sacklike molecules. It is called a peptidoglycan or murein (latin murus,

wall). The structure and enzymatic biosynthesis of the peotidoglycan were largely

elucidated during investigations on the mechanism of action of the antibiotic

penicillin by J. Strominger and his colleagues. The basic recurring unit in the

peotidoglycan structure is the muropeptide. It is a disaccharides of N-acetyl-D-

glucosamine and N-acetylmuramic acid in β ( 4) linkage. The backbone may

be regarded as a substituted chitin with D-lactic acid substituted on alternating

residues. To the carboxyl group of the N-acetylmuramic acid residues of the

backbone are attached tetrapeptide side chains each containing L-alanine, D-alanine,

D-glutamic acid or D-glutamine, and either meso-diaminopimelic acid, L-lysine, L-

hydroxyllysice, or ornithine depending on the bacterial species.

The parallel polysaccharide chains of the cell wall are cross-linked through their

peptide side chains. The terminal D-alanine residue of the side chain of one

polysaccharide chain is joined covalently with the peptide side chain of an adjacent

polysaccharide chain, either directly, as in E. coli, or through a short connecting

peptide, e.g., the pentaglycine in Staphylococcus aureus. The peptidoglycan forms a

126

completely continuous, covalent structure around the cell; Gram-positive bacteria are

encased by up to 20 layers of cross-linked peptidoglycan.

The peptidoglycan structure of the bacterial cell wall is resistant to the action of

peotide-hydrolyzing enzymes, which do not attack peptides containing D-amino

acids.

In addition to the peptidoglycan framework, bacterial cell walls contain a number of

accessory polymers, which make up almost 50 percent of the weight of the wall.

These accessory components differ from one species to the another. There are three

types of accessory polymers: (1) teichoic acids, (2) polysaccharides, and (3)

polypeptides or proteins.

The walls of Gram-negative cells, such as E. coli, are much more complex than those

of Gram-positive cells. Their accessory component consist of polypeptides,

lipoproteins, and particularly, a very complex

lipopolysaccharide whose structure is just

beginning to be understood. It has a

trisaccharide backbone repeating unit, consisting

of two heptose (seven-carbon) sugars and

octulosonic acid (an eight carbon sugar). To this

backbone are attached oligosaccharide side

chains and the fatty acid β-hydroxymyristic

acid, which gives this complex structure its lipid

character. The lipopolysaccharide forms an

outer lipid membrane and contributes to the

complex antigenic specificity of Gram-negative

cells. The cell wall of Gram-positive organisms

can be tough of as a rigid, brittle box, like the

127

shell of a crustacean, whereas the cell wall of Gram-negative organisms has an outer

lipid-rick skin, with the rigid peptidoglycan skeleton buried underneath.

1.5 STORAGE POLYSACCHARIDE

These polysaccharides, of which starch is the most abundant in plants and glycogen

in animals, are usually deposited in the form of large granules in the cytoplasm of

cells. Glycogen or starch granules can be isolated from cell extracts by differential

centrifugation. In times of glucose surplus glucose units are stored by undergoing

enzymatic linkage to the ends of starch of glycogen chains; in times of metabolic

need they are released enzymatically for use as fuel.

1.5.1 STARCH

Starch occurs in two forms, α -amylose and amylopectin. α -Amylose consists of

long unbranched chains in which all the D-glucose units are bound in α (14)

linkages. The chains are polydisperse and vary in molecular weight from a few

thousand to 500,000. Amylose is not truly soluble in water but forms hydrated

micelles, which give a blue color with iodine. In such micelles, the polysaccharide

chain is twisted into a helical coil. Amylopectin is highly branched; the average

length of the branches is from 24 to 30 glucose residues, depending on the species.

The backbone glycosidic linkage is α (14), but the branch points are α (16)

linkages. Amylopectine yields colloidal or micellar solutions, which give a red-violet

color with iodine. Its molecular weight may be a high as 100 million.

The major components of starch can be enzymatically hydrolyzed in two different

ways. Amylose can be hydrolyzed by α α(14)]-glucan 4-

glucanohydrolase], which is present in saliva and pancreatic juice and participates in

the digestion of starch in the gastrointestinal tract. It hydrolyzes α(14) linkages at

random to yield a mixture of glucose and free maltose; the latter is not attacked.

128

Amylose can also hydrolyzed by β α(14)-glucan maltohydrolase]. This

enzyme, which occurs in malt, cleaves away successive maltose units beginning

from the nonreducing end to yield maltose quantitatively. The α- and β- amylases

also attact amylopectine. The polysaccharides of intermediate chain length that are

formed from starch components by the action of amylases are called dextrins.

Neither α- nor β-amylases can hydrolyze the α (16) linkages at the branch points

of amylopectine. The end product of exhaustive β -amylase action on amylopectin is

a large, highly branched core, or limit dextrin, so called because it represents the

limit of dextrin, so called because it represents the limit of the attack of β -amylase.

A debranching enzyme [α (16)-glucan 6-glucanohydrolase, also called α (16)-

glucosidase] can hydrolyze the α (16) linkages at the branch points. The combined

action of a β -amylase and an α(16)-glucosidase can therefore completely degrade

amylopectin to maltose and glucose.

1.5.2 GLYCOGEN

Glycogen is the main storage polysaccharide of animal cells, the counterpart of

starch in plant cells. Glycogen is especially abundant in the liver, where it may attain

up to 10 percent of the wet weight. It is also present to about 1 to 2 percent in

skeletal muscle. In liver cells the glycogen is found in large granules, which are

themselves clusters of smaller granules composed of single, highly branched

molecules with a molecular weight of several million.

Like amylopectin, glycogen is a polysaccharide of D-glucose in α (14) linkage.

However, it is a more highly branched and more compact molecule than

amylopectine; the branches occur about ever 8 to 12 glucose residues. The branch

linkages are α (16). Glycogen can be isolated from animal tissues by digesting

129

them with hot KOH solutions, in which the nonreducing α (14) and α (16)

linkages are stable. Glycogen is readily hydrolyzed by α - and β -amylases to yield

glucose and maltose, respectively; the action of β -amylase also yields a limit

dextrin. Glycogen gives a red-violet color with iodine.

1.5.3 OTHER STORAGE POLYSACCHARIDES

Dextrans, too, are branched polysaccharides of D-glucose, but they differ from

glycogen and starch in having backbone linkages other than α (14). Found as

storage polysaccharides in yeasts and bacteria, they vary in their branch points,

which may be 12, 13, 14, or 16 in different species. Dextrans form highly

viscous, slimy solutions. Fractans (also called levans) are homopolysaccharides

composed of D-fructose units; they are found in many plants, Inulin, found in the

artichoke, consists of D-fructose residues in β (21) linkage. Mannans are mannose

homopolysaccharides found in bacteria, yeasts, molds, and higher plants. Similarly,

xylans and arabinanas are homopolysaccharides found in plant tissues.

1.6 STRUCTURE AND FUNCTION OF MUCOPOLYSACCHARIDES

OR GLUSOSAMINOGLYCANS

The acid mucopolysaccharides are a group of related heteropolysaccharides usually

containing two types of alternating monosaccharide units of which at least one has an

acidic group, either a carboxyl or sulfuric group. When they occur as complexes with

specific proteins, they are called mucins or mucoproteins; in this class of

glycoproteins the polysaccharide makes up most of the weight. Mucoproteins are

jellylike, sticky, or slipery substances; some provide lubircation, and some function

as a flexible intercellular cement.

130

Table 10-2 Acid mucopolysaccharides

Polysaccharides Constituents Occurrence

Hyaluronic acid Glucuronic acid, N-acetyl-D-glucosamine Synovial

fluid

Chondroitin Glucuronic acid, N-acetyl-D-glucosamine Cornea

Chondroitin 4-

sulfate

Glucuronic acid, N-acetyl-D-glucosamine 4-

sulfate

Cartilage

Dermatan sulfate Iduronic acid, N-acetyle-D-galactosamine 4-

sulfate

Skin

Keratan sulfate Galactose, galactose 6-sulfate, N-acetyl-D-

glucosamine 6-sulfate

Cornea

Heparin Glucosamine 6-sulfate, glucuronic acid 2-

sulfate, iduronic acid

Lung

The most abundant acid mucopolysaccharides is hyaluronic acid, present in cell

coats and in the extracellular ground substance of the connective tissues of

vertebrates; it also occurs in the synovial fluid in joints and in the vitreous humor of

the eye. The repeating unit of hyaluronic acid is a disaccharide composed of D-

glucuronic acid and N-acetyl-D-glucosamine in β(3) linkage. Since each

dissacharide unit is attached to the next by β (4) linkages, hyaluronic acid contains

alternating β (3) and β (4) linkages. Hyaluronic acid is a linear polymer.

Because its carboxyl groups are completely ionized and thus negatively charged at

pH 7.0, hyaluronic acid is soluble in water, in which it forms highly viscous

131

solutions. The enzyme hyaluronidase catalyzes hydrolysis of the β (4) linkages of

hyaluronic acid; this hydrolysis of accompanied by a decrease in viscosity.

Another acid mucopolysaccharide is chondroitin, which is nearly identical in

structure to hyaluronic acid; the only difference is that it contains N-acetyl-D-

galactosamine instead of N-acetyl-D-glucosamine residues. Chondroitin itself is only

a minor component of extracellular material, but its sulfuric acid derivatives,

chondroitin 4-sulfate (chondroitin A) and chondroitin 6-sulfate (chondroitin C), are

major structural components of cell coats, cartilage, bone, cornea, and other

connective-tissue structures in vertebrates. Dermatan sulfate and keratan sulfate are

acid mucopolysaccharides found in skin, cornea, and bony tissues. A related acid

mucopolysaccharide is heparin, which prevents coagulation of blood, It is found in

the lungs and in the walls of arteries.

Recently it has been discovered that some acid mucopolysaccharides contain the

element silicon, which is essential in the nutrition of rats and chicks. The silicon is

bound to the polysaccharides in covalent form.

1.7 CARBOHYDRATES OF GLYCOPROTEINS AND GLYCOLIPIDS

1.7.1 GLYCOPROTEINS

Among the several different classes of conjugated proteins, the glycoproteins, which

contains carbohydrate groups attached covalently to the polypeptide chain, represent

a large groups of wide distribution and considerable biological significance. In fact,

on closer study many proteins once thought to be simple proteins, i.e., containing

only amino acid residues, have been found to contain carbohydrate groups. The

percent by weight of carbohydrate groups in different glycopoteins may vary from

less than 1 percent in ovalbumin to as high as 80 percent in the mucoproteins.

Glycoproteins having a very high content of carbohydrate are called proteoglycans.

132

Glycoproteins are found in all

forms of life. In vertebrates most

but not all of the glycoproteins

are extracellular in occurrence and function or are secreted from cells; it has

accordingly been suggested that one pupose of the attached sugar residues is to label

the protein for export from the cell. Among the glycoproteins having extracellular

location or function are the cell-coat glycoproteins, the blood glycoproteins, the

cirulating forms of some protein hormones, the antibodies, various digestive

enzyems secreted into the intestine, the mucoproteins of mucous secretions, and the

glycoproteins of extracellular basement membranes.

Many different monosaccharides and monosaccharide derivatives have been found in

glycoproteins. The linear or branched side chains of glycorproteins may contain from

two to dozens of monosaccharide residues, usually of two or more kinds. Often the

terminal monosaccharide unit is a negatively charged of N-acetylneuraminic acid, a

sialic acid.

The oligosaccharide groups of most glycoproteins are convalently attached to the R

groups of specific amino acid residues in the polypeptide chain. Three different types

of linkages have been found. In some glycoproteins, e.g., ovalbumin and the

immunoglobulins, the oligosaccharide is attached via a glycosylamine linkage

between N-acetyl-D-lucosamine of the bligosaccharide to the amide nitrogen of an

asparagine residue in the polypeptide chain. In a second class of glycoproteins,

including the submaxillary mucoprotein, there is a glycosidic bond between N-

acetyl-D-galactosamine of the side-chain oligosaccharide and the hydroxyl group of

a serine or threonine residue. The submaxillary mucins have recurring units of about

28 amino acid residues; each redcurring unit contains theree oligosaccharide side

chains. In the third class of glycoproteins, respresented by collagen, the

oligosaccharide side chains are attached to the hydroxyl groups of hydroxylysine

133

residues. The precise sequences of residues in the oligosaccharide side chains are

known in only a few cases.

The antifreeze proteins found in the blood plasma of Antarctic fishes are particularly

interesting. Their backbones consist of the recurring amino acid sequence Ala-Ala-

Thr; the disaccharide galactosy-N-acetyl-galactosamine is attached to every

threonine residue. The molecular weights of these proteins vary from 10,000 to

23,000. The antifreeze proteins have a flexible, expanded structure in water which

presumably interferes with the formation of the crystal lattice of ice.

The human blood-group proteins contain oligosacchride side chins with residues of

L-fructose, D-galactose, N-acetyl-D-galactosamine, and N-acetyl-D-glucosamine;

these side chains determine the glood-group specificity.

Table - Some glycoproteins, grouped according to biological occurrence. Note that

most glycoproteins are extracellular.

Blood plasma

Fetuin

a1-Acid glycoprotein

fibrinogen

Immune Globulins

Thyroxine-binding protein

Blood-group proteins

Urine

Urinary glycoprotein

Hormones

Chorionic gonadotrophin

Follicle-stimulating hormone

Thyroid-stimulating hormone

134

Enzymes

Ribonuclease B

b-Glucuronidase

Pepsin

Serum cholinesterase

Egg white

Ovalbumin

Avidin

Ovomucoid

Mucus secretions

Submaxillary glycoproteins

Gastric glycoproteins

Connective tissue

Collagen

Cell membranes

Glycophorin of erythrocyte membrane

Extracellular Membranes

Basement-membrane glycoprotein

Lens-capsule glycoprotein

1.7.2 GLYCOLIPIDS

These are compounds containing a fatty acid, a carbohydrates, a complex alcohol,

and nitrogen, but no phosphorus.

Glycolipids, galactolipids or

cerebrosides occur in considerable

amounts in the white matter of the

135

brain and of all nervous tissue. They usually occur in the amorphous state, but are

also known as liquid crystals. They are insoluble in ether but soluble in hot alcohol.

On hydrolysis, they give a fatty acid, sphingosinol, and a sugar, usally glactose.

There are four individual memebers of this group which differ only in the nature of

their fatty acids, The general structural formula for galactolipids is as below.

The sphingosin-fatty acid moieiy is called a ceramide. The nature of the fatty acid

radical (R.CO) in the four galactolipids is given in the following table.

Name of the lipid Fatty acid radical (R.CO -) Name of the fatty acid

Cerasin CH (CH2)22 COOH Lignoceric acid

Phrenosin

(cerebron)

CH3 (CH2)22 CHOH.CHOH A Hydroxylignoceric

(phrenosinic) acid

Nervone CH3

(CH2)7.CH=CH.(CH2)12.COOH

Nervonic acid

Hydroxy nervone C24H46O3 Hydroxynervonic acid

In addition to the above compound lipids there are globosides, hematosides and

gangliosides. There are structurally similar to glycosides with the only difference

that in the former the sugar residues are acetylated amino sugars, e.g. D-

galactosamine, in the middle one they are sialic acid while in the latter acetylated

amino sugars and sialic acid both are present.

1.8 ROLE OF SUGARS IN BIOLOGICAL RECOGNITION BLOOD GROUP

SUBSTANCES- VITAMIN C (ASCORBIC ACID)

Ascorbic acid is a derivative of carbohydrates. It possesses an asymmetric carbon

atom marked by *) and is therefore optically active. It exists in the following two

forms.

136

L-Ascorbic acid and L-dehydroascorbic acid are the only known naturally occurring

biologically active substances, the corresponding D-forms are generally inactive.

It is a white crystalline solid freely soluble in water, its most important chemical

property is its powerful reducing activity during which it gives up its two hydrogen

atoms and is oxidised to dehydroascorbic acid. This oxidation of ascorbic acid in the

body is reversible and affected by the – SH group of glutathione. Howerver, ascorbic

acid may be irreversibly oxidised beyond the stage of dehydroascorbic acid to oxalic

acid via deketagulonic acid.

The stability of ascorbic acid is affected by the pH, temperature,, contact with

oxygen and traces of metals especially copper. It is rapidly oxidised in the presence

of light, oxygen and traces of coppeer at pH above4, expecially if the solution is

warmed. Thus ascorbic acid is liable to be destroyed during preparation and cooking

of many foods. Moreover, fruits and vegetables contain an enzyme, ascorbic acid

oxidase, which accelerates the oxidation of ascorbic acid in the presence of oxygen.

The vitamin is distributed both in plant and animal kingdoms. In plant community

the important sources are leaves and flowers (e.g., rose hips, pine needles), fruit (e.g.,

lemons oranges, black currants, grapefruit, guava, gooseberries, strawberries, apples,

bananas, etc) and green vegetables (e.g. cauliflowers, cabbage, green peas, beans,

tomatoes, etc.) Because of the chemical instability of ascorbic acid, much of it is

137

destroyed in the preparation of food. In animal, the vitamin occurs in tissue and

various glands or organs, e.g. adrenal gland, thymus, pituitary, corpus luteum, liver,

lung and heart muscle. Milk, potatoes and blood also contain small quantity of

ascorbic acid.

In plants, mostly, the vitamine occurs as such but in animsl and some plants it is

found in equilibrium with dehydroascorbic acid. Besides these two forms ascorbic

acid is also found in the combined form (ascorbigen).

Ascorbic acid is absorbed readily from the small intestice, peritoneum, and

subcutaneous tissue. Although the human body has reserve stores of the vitamin,

there is no evidence to show that any particular organ or tissue serves this function.

Under normal dietary conditions, about 25-50 % of the ingested vitamin is excreted

in the urine; the rest being degraded to diketogulonic acid and oxalic which are

excreted in the urine. It is also secreted in the milk.

Function: The biological activity of this vitamin is due to its reversible oxidation.

It has been seen that there are special enzymes or compounds which help in the

oxidation and reduction of ascorbic acid; e.g., glutathione reduces the oxidised form

of vitamin C; whereas some purines (xanthine, uric acid, theophyline etc.) protect the

vitamin against oxidation. On the other hand, the enzyme ascorbic acid oxidase

brings about the oxidation of ascorbic acid.

On the basis of the above behavious or ascorbic acid, Szent-Gyorgyl suggested that

the vitamin takes part in the respiratory system according to the follwing reactions.

Ascorbic acid + O2 Dehydro-ascorbic acid + H2O ..i

Flavone + H2O Oxidised flavone + H2O ..ii

Oxidized flavone + Ascorbic acid Dehydroascorbic acid + flavone ..iii

Dehydroascorbic acid + glutathione

Ascorbic acid + Oxidized glutathione … iv

138

Oxidized glutathione + Glucose phosphate

Glutathione + H2O2 + H2O … v

In the absence of the compounds such as flovones the reactions (ii) and (iii) are

replaced by

H2O2 H2O + ½ O2

Ascorbic acid is also found to be linked with the amino acid metabolism.

1.9 CARBOHYDRATE METABOLISM

.AN OVERVIEW OF RESPIRATION.

Check your progress – 1

1. Note : - Write your answer in the space given

2. Compare your answer with the one given at the end of the unit.

1. What are carbohydrates? What is their functions significance.

2. Write one function of : -

3. Deoxy sugars, N-acitylmeramic acid, sialic acid & cellulose, Glycogen.

4. What are muco polysaccharides.

-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

139

a) You must bear a clear understanding in mind that both photosynthesis and

respiration involves gaseous exchange but light reaction of photosynthesis

requires sunlight whereas respiration occurs all the time.

O2 is utilized in the process &Co2 is released.

b) The sites of respiration are cytoplasms and mitochondria. The organic

compounds are broken down inside the cells by oxidation process, known as

cellular respiration. The energy released is stored in pyrophosphate bonds of

ATP.

ADP + H3PO4 ATP(ADP˜P)

Energy stored in ATP is utilized for carying out different cellular and biological

activites because of this, energy is called energy currency of the cell.

c) The overall reaction is as follows:

C6H12O6 + 6O2 + 38ADP+38iP 6CO2 + 6H2O + 38ATP

The main features of respiration are:

Oxidation of organic compounds occurs in under aerobic conditions

Complete oxidation occurs

End products are CO2 & H2O

Higher amount of (673 Kcal )energy is liberated out

Process occurs in cytoplasm and mitochondria

Various respiratory substance are: glucose, fructose, fats, protein, etc.

The ratio of volume of CO2 released to the volume of O2 absorbed during

respiration is called respiratory ratio or R.Q.

Volume of CO2 released

R.Q. =

140

Volume of O2 absorbed

To develop a clear understanding of the process let us understand the mechanism of

respiration

MECHANISM OF RESPIRATION

Cellular respiration is a complicated process which is completed in many steps. for

every step, a particular enzyme is required which works in a sequential manner one

after the another.

it is completed in 3 steps:

d) Glycolysis / EMP pathway

e) Oxidation of pyruvic acid

f) ETC & oxidative phosphorylation

1.9.1 GLYCOLYSIS/ EMP PATHWAY

Greek, glucose – sugar, lysis – dissolution. If I say that glycolysis is a fermentive

pathway would you agree?

Reasons to support my statement are:

d) It does not involves O2 intake

e) ATP generated is through substrate level phosphorylation.

f) Organic compound donates electrons and organic compound accepts

it.

This process was discovered by three German scientists Embden, Meyernhof and

Parnas. On their name the pathway is also called EMP pathway.

All the reactions of glycolysis take place in the cytoplasm and

through the glycolysis glucose is oxidized into pyruvic acid in presence of many

141

enzymes present in the cytoplasm. Thus the process of sequential oxidation of

glucose into pyruvic acid is known as glycolysis.

Energy

production

during

glycolysis:

During

glycolysis process two molecules of ATP are utilized to convert glucose into

glucose-6-PO4 & fructose -1, 6 diphosphate wherea 4 molecules of ATP and 2

molecules of NADH2 are produced during following steps.

142

(One molecule of NADH2 gives three molecules of ATP by ETC)

Total production of ATP in glycolysis cycle

Reaction number No. of ATP molecule produced

(vii) 1,3 –DPG-Ald 1,3-DPGA 2NADH2(2*3) = 6ATP

(viii)1,3 – DPGA 3-PGA 2ATP = 2ATP

(xi) PEPA Pyruvic Acid 2ATP = 2 ATP

10ATP

As 2 molecules of ATP are utilized during glycolysis, thus net gain of ATP

molecules during this process is 8 molecules of ATP

10 ATP – 2 ATP

Net gain of ATP = 8 ATP

SIGNIFICANCE OF GLYCOLYSIS:

a) Generates ATP

b) Precursor metabolic generation

c) Generates reducing power

Main enzymes are:

1) phosphofructokinase

2) pyruvate kinase

3) pyruvate enol carboxylase

143

General patter of metabolism leading to synchronization in Ecoli cells

Role of ATP :

Adenosine - P ~ P ~ P + H2O adenosine - P~P + P 4° = -7.8Kcal

Adenosine - P ~ P + H2O adenosine ~P + P 4° = 7.3Kcal

Adenosine ~ P + H2O adenosine + P 4° =3.4Kcal

High energy compounds other than ATP

Compound cause action in Priosyn.of:

GTP Protein(ribosome function)

CTP Phospholipids

UTP Peptidoglycan layer of bacterial wall

Dcoxythymidine~ P~P~P lipopolysaccarid layer of bacterial wall

dTTTP

Acyl~SCoA Fatty acids

OXIDATION OF PYRUVIC ACID

The fat of pyruvic acid produced during glycolysis depends on whether oxygen is

available or not

A) In case of anaerobic condition it is used as hydrogen acceptor for the two

molecules of NADH generated during glycolysis and is converted into lactic acid.

Alcoholic fermentation of pyruvic acid in plants: in yeast cells anaerobic oxidation

of pyruvic acid takes place as follows:

Decarboxylation of pyruvic acid in presence of pyruvic decarboxylase enxyme to

produced acetaldehyde.

CH3COOH Pyruvic Decarboxylase CH3CHO + O2

144

1) In presence of alcohol dehydrogenase enzyme acetaldehyde reacts with

NADH2 to produce ethyl alcohol and NAD.

2CH3.CHO+2NADH2 A.dehydrogenase CH3.CH2.OH + 2NAD

Acetaaldehyde Ethylalcohol

In animal cells lactic acid is formed

B) Aerobic oxidation of pyruvic acid according to Wood et.al.(1942) and H.A.Kreb‘s

(1943) in the presence of O2 oxidation of pyruvic acid takes place through Kreb‘s

cycle or T.C.A cycle.

Before entering Kreb‘s cycle pyruvic acid gets decarboxylated to produce acetyl-

CoA which enters the Kreb‘s cycle and oxidize to produce CO2 . H2O and ATP.

Pyruvic Decarboxylase

Pyruvic acid + Coenzyme A + NAD Acetyl-CoA +NADH2

C) Fate of pyruvic acid to alanine during amino acid synthesis pyruvic acid react

with glutamic acid alanine.

Pyruvic acid + Glutamic acid Alanine +α-Keto glutaric acid

1.9.2 T.C.A. CYCLE/KREB’S CYCLE:

This cycle was described for the first time by H.A.Kreb in 1943. It is also known as

T.C.A. cycle because it produces tricarboxylic acids the process completes in

mitochondrial crests.

145

Diag

ram of Mitochondria

All the chemical reaction of Kreb‘s cycle can be summarized in following steps:

146

10. Aerobic oxidation of P.A

11. Condensation of Acetyl-CoA with oxalo-acetic acid

12. Isomerisation of citric acid into isocitric acid ,{(a) dehydration and (b)

hydration)}

13. Oxidative decarboxylation of isocitric acid (a) dehydration and (b)

decarboxylation)

14. Oxidative decarboxylation of α-Keto glutaric acid.

15. Conversion of succinyl CoA into succinic acid.

16. Dehydrogenation of succinic acid into fumaric acid

17. Hydration of fumaric acid into malic acid

18. Dehydrogenation of malic acid in OAA.

Overall reaction of respiration is:

Glycolysis + Kreb‘s cycle = Glucose + 4ADP + 4H3PO4+ 8NAD++ NADP

+ +2FAD

6CO2 + 4 ATP + 8NADH + 10H+ +2NADPH + 2FADH2

Thus as a result of oxidation of pyruvic acid, one molecule of CO2 in oxidative

decarboxylation and two molecules of CO2 in Kreb‘s cycle are liberated. The total

number of CO2 evolved becomes 3 which indicates that 3 carbon pyruvic acid has

been completely oxidized in glycolysis.

Because two molecules of P.A. which are formed by one molecule of glucose in

glycolysis, enter into Kreb‘s cycle for oxidation, a total of 6CO2 molecule will be

evolved.

2PA * 3CO2 = 6CO2

All the NADH2 and FADH2 are oxidized to NAD and FAD through a chain of

reaction c/a etc.in this process ATP molecules are released (1NADH2 = 3ATP,

1FADH2 = 2ATP). In the process of Kreb‘s cycle 8 molecules of NADH2 =24ATP ,

2FADH2 = 4 ATP and two molecules of ATP are synthesized from 2GTP.

ELECTRON TRANSPORT SYSTEM AND OXIDATIVE PHOSPHORYLATION

147

Electron Transport System (ETC)

During repiration simple carbohydrates and intermediate compounds like

phosphoglyceraldehyde, pyruvic acid, isocitric acid, α ketoglutaric acid, succinec

acid and malie acid are oxidized. Each oxidative step involves release of a pair of

hydrogen atoms which dissociates into two protons and two electrons.

2H 2H+ + 2e

-

These protons and electrons are accepted by various hydrogen acceptors like

NAD,NADP, FAD etc. After accepting hydrogen atoms these acceptors get reduced

to produce NADH2, NADPH2 and FADH2. The pairs of hydrogen atoms released a

series of coenzymes and cytochromes which form electron transport system, before

reacting with O2 to form H2O.

½ O + 2H+ + 2e

- H2O

2NADH + O2 + 2H+ 2NAD

++ 2H2O

As you know that H ions and electrons removed from the respiratory substrate

during oxidation do not directly react with oxygen. Instead they reduce acceptor

molecules NAD and FAD to NADH2 and FADH2. These molecules then transfertheir

electron to a system of electron acceptors and transfer molecules. The proteins of the

inner mitochondrial membrane act as electron transporting enzymes. They are

arranged in an ordered manner in the membrane and function in a specific sequence.

This assembly of electron transport enzymes is known as mitochondrial respiratory

chain or the electron transport chain. Specific enzymes of this chain receive electrons

from reduced prosthetic groups, NADH2 or FADH2 produced by glycolysis and the

TCA cycle. The electrons are then transported successively from enzyme to enzyme,

148

down a descending ‗stairway‘ of energy yielding reactions. This process takes place

in mitochondrial cristae which contain all the components of E,T.S.

Components of electron transport system: the electron transport system is made

up of following enzymes and proteins:

10. Nicotinamide adenine dinucleotide (NAD).

11. Flavoproteins (FAD and FMN).

12. Fe-S protein complex.

13. Co-enzyme Q or ubiquinone.

14. Cytochome-b

15. Cytochrome-c1.

16. Cytochrome-c

17. Cytochrome-a

18. Cytochrome-a3.

All the above enzymes are found in F1 particles of mitochondria.

Mechanism of action of electron transport system: During respiration electron

pairs liberated from respiratory compounds are accepted by coenzymes like NAD

or NADP and FMN etc. The transfer of electrons in all compounds except

succinic acid takes place first in NAD+ or NADP

+ and later on in FAD. The

transfer of electgrons from succinic acid takes place diretly to the FAD and not

through NAD+ or NADP

+. Due to this reason only two molecules of ATP are

formed in the formation of fumaric acid from succinic acid whereas in case of

other compounds 3 ATP molecules are produced because these cases the

electrons are first picked up by NAD.

Different Steps of E.T.S. are as follows:

149

3. Hydrogen pairs released from different substrates of Krebs cycle except

succinic acid reacts with NAD+. The electrons and proton are transferred to

NAD causing its reduction and one proton is released in the medium.

2H 2H+ + 2e

-

(protons) (electrons)

NAD + 2H+ + 2e

- NADH + H

+

(reduced) (ion pool)

4. Now, 2e- and one H

+ are transferred from NADH to FAD causing oxidation

of NADH to NAD and reduction of FMN into FMNH2. One H+ is picked

up from hydrogen ion pool to complete this reaction.

The free energy released at this step is stored during oxidative phophorylation

and one molecule of ATP is generated fronm ADP and inorganic phosphate.

The hydrogen pair from succinic acid is first transferred to FAD to form

FADH2. The FADH2 transfers electrons to coenzyme Q throught Fe-S and CoQ. The

electrons pass to cytochromes Cyt-b, Cyt-c1, Cyt-c, Cyt-a, Cyt-a3 and then to oxygen

atoms. Oxygen atom accepts those electrons and reacts with hydrogen ions of the

matrix to form water.

O2 + 4e- 2(O

--)

2(O--) + 4e

+ 2H2O

Oxygen is thus the terminal electron acceptor of the mitochondrial respiratory

chain.

At each step of electron acceptor has a higher electron affinity than the electron

donor from which it receives the electron. The energy from such electron transport is

utilized in transporting protons from the matrix across the inner membrane to its

outer side. This creates a higher proton concentration outside the inner membrane

150

than in the matrix. The difference in proton concentration across the inner membrane

is called proton gradient.

The reduction of various cytochromes requires only electrons and no protons.

Each cytochromes possesses an iron elements in the centre which functions for

accepting (Fe3+

Fe2+

) or donating (Fe2+

Fe3+

) When a cytochrome accepts electrons, it

is reduced and if it donates electrons, it is oxidized.

Oxidative Phosphorylation

In all living beings ATP generated during oxidative breakdown of complex food

products. This process of synthesis of ATP molecules from ADP and inorganic

phosphate by electron transport system of aerobic respiration called as oxidative

phosphorylation.

ADP + iP O

2 ATP

E.T. Chain

The process of oxidative phosphorylation takes place in mitochondrial crests through

electron transport chain.

Due to high proton concentration outside the inner membrane, protons return

to the matrix down the proton gradient. Just as a flow of water from a higher to lower

level can be utilized to turn a water-wheel or a hydroelectric turbine, the energy

released by the flow of protons down the gradient is utilized in synthesizing ATP.

The return of proton occurs through the inner membrane particles. In the F0-F1

complex the F1 head piece functions as ATP synthetase. The latter synthesizes ATP

from ADP and inorganic phosphate using the energy from the proton gradient.

Transport of two electrons from NADH2 by the electron transport chain

simultaneously transfers three pairs of protons to the outer compartment. One high

energy ATP bond is produced per pair of protons returning to the matrix through the

151

inner membrane particles. Therefore, oxidative phosphorylation produces three ATP

molecules per molecules of NADH2 oxidized. Since FADH2 donates its electrons

further down the chain. Its oxidation can only produce two ATP molecules.

During oxidative phosphorylation ATP molecules are produced during following

steps:

IV. When NADH2 is oxidized to NAD by reacting with FAD.

V. When the electron transfer from cytochrome-b to cytochrome-c1.

VI. When the electron transfer from cytochrome-a to cytochrome-a3.

Now it is clear that oxidation of one molecule of reduced NADH2 or NADPH2

results in the formation of 3 molecules of ATP while oxidation of FADH2 leads to

the formation of 2 molecules of ATP.

1.9.3

PENTOSE

PHOSPHATE PATHWAY : AN ALTERNATIVE PATHWAY FOR

GLUCOSE BREAKDOWN.

152

Warberg et al (1935) & Dicken‘s suggested an alternative oxidative pathway for

glucose oxidation. It is named as Pentose Phosphate Pathway or Hexose

Monophosphate shunt.

Out of 6 only molecule of glucose monophosphatic is oxidized into CO2 in each

cycle of P.P.P & 5 molecules of fructose monophosphatic & glucose

monophosphatic are formed.

Total 12 NADPH2 molecules are formed in each cycle which are oxidized by

cytochrome cycle into 12 molecules of NADP. Total 36 ATP molecules are

produced during whole process.

Significance of P.P.P.

1. It is substitute for glycolysis and Kreb‘s cycle.

2. 5-carbon compounds produced are used in the synthesis of nucleic acids.

153

3. This

pathway can supply required quantity of the energy to the cell, when

glycolysis & Kreb‘s cycle do not occur due to some reason.

154

CHECK YOUR PROGRESS 2

1. Note : - Write your answer in the space given

2. Compare your answer with the one given at the end of the unit.

1. What is EMP Pathway? How many ATP are generated through it

2. Name of end product of

a. Glycolysis b. Kreb‘s Cycle c. Glycogenesis

---------------------------------------------------------------------------------------------------------

---------------------------------------------------------------------------------------------------------

---------------------------------------------------------------------------------------------------------

---------------------------------------------------------------------------------------------------------

---------------------------------------------------------------------------------------------------------

---------------------------------------------------------------------------------------------------------

---------------------------------------------------------------------------------------------------------

---------------------------------------------------------------------------------------------------------

---------------------------------------------------------------------------------------------------------

---------------------------------------------------------------------------------------------------------

---------------------------------------------------------------------------------------------------------

---------------------------------------------------------------------------------------------------------

---------------------------------------------------------------------------------------------------------

---------------------------------------------------------------------------------------------------------

---------------------------------------------------------------------------------------------------------

---------------------------------------------------------------------------------------------------------

-----------------------------

---------------------------------------------------------------------------------------------------------

--------

155

1.9.4 GLYCOGEN SYNTHESIS - STORAGE OF CARBOHYDRATES-

Glycogen synthesis and breakdown. In the absence of urgent physiological

demands for oxidative energy or conversion to special products. Excess of glucose is

stored in the form of glycogen in the liver and other tissues: the process of

biosynthesis of glycogen from glucose (or other sugars) is known as glycogenesis.

Glycogen is synthesized in practically all the tissues of the body but the major sites

are liver and muscles. The storage of glucose in the form of glycogen, which is

converted back to glucose, as the time of requirement, is very important because in

the absence of this the tissues would be flooded with excess of glucose immediately

after mean and starved of it at all other times. The carbohydrate reserve in the adult

of it at all other times. The carbohydrate reserve in the adult human liver (1.8 kg) is

about 100 gm and since accumulation of such a large amount of the smaller molecule

like glucose will give concentration of glucose inside the liver cells will nearly be

doubled leading to disastrous results. It is, therefore, advantageous to the organism to

store its glucose in the form of a polymer, glycogen, which has a high molecular

weight and correspondingly low osmotic pressure. In case carbohydrate rich diet is

taken, the liver tissue may immediately store glycogen about 5-6% of its weight. The

liver glycogen may be exhausted after a fast of 12-18 hours. Other important store of

glycogen is muscles which may contain about 0.7-1.0% glycogen and since an adult

human has about 35 kg. of muscles. Muscle glycogen is utilised in case of severe

body exercise or when the liver glycogen is completely exhausted. Now since the

amount of glycogen which can be stored in the body is limited, excess quantities of

glucose are converted to fatty acids and stored as triglycerides (fat).

Glycogenesis Pathway

156

Although synthesis of glycogen from glucose can occur in most of the tissues of the

body; liver and muscles are the most important sites. Further, liver is the only viscera

which can synthesize glycogen from monosaccharides other than glucose. However,

the biochemical reactions for the polymerization of glucose to glycogen in liver and

muscles are found to be similar.

The various steps involved in glycogenesis are described here.

1. Phosphorylation of glucose. First of all glucose1 is phophorylated (activated)

by A.T.P. in presence of the enzyme hyxokinase or more specifically

glucokinase and Mg++

(activator) to glucose-6-phosphate. Both of these

enzymes are found to be present in the high mammals.

Glucose + ATP Glucose-6-phosphate + ADP

The enzyme glucokinase (hexokinase) is inhibited by adrenal cortical

hormones (gluco-corticoids) and this inhibition is facilitate by anterior pituitary

hormones. However, the inhibition is removed by insulin. It is important to note that

formation of glucose-6-phosphate acts as a locking mechanism to keep the glucose

within the cell since it is not permeable to cell membrane while glucose is readily

permeable.

2. Conversition of glucose-6-phosphate to glucose-1-phosphate. Glucose-6-

phosphate is reversibly transferred to glucose-1-phosphate in presence of

phosphoglucomutase; this reaction requires the presence of glucose-1, 6-

diphosphate as coenzyme.

Glucose –6-P Glucose-1-P

3. Conversion of glucose-1-phosphate to UDPG. The glucose 1-phosphate now

combines with uridine triphosphate (UTP) in presence of uridine diphosphate

glucose pyurophosphorylase (UDPG pyrophosphorylase) to form uridine

diphosphoglucose (UDPG) with the elimination of pyrophosphate2 (P~P).

157

Glucose-1-P + UTP UDPG + Pyrophosphate (P~P)

(converted into H3PO4)

4. Conversion of UDPG to glycogen. The uridine diphosphoglucose molecule

now transfers its glucose molecules to the non-reducing outer end of an

existing glycogen chain under the influence of the glycogen-UDP glucosyl

transferas (commonly called glycogen synthetase); one glucose unit is thus

added to a primer glycogen via 1-4 linkage. This reaction is driven forward

when excess of glucose –6-phosphate is present. The UDP formed as a by-

product is converted to UTP is presence of ATP.

UDPG + Glycogen primer (1, 4-Glucosylunit)x + UDP

UDP + ATP UDP + ADP

Glycogen synthesis is found to exist in two forms namely synthesis-D

(dependent) which is active only in presence of glucose-6-phosphate and

synthetase –I (independent) which is independent of the concentration of

glucose –6-phosphate and is the active enzyme for all practical purposes.

Insulin favours the conversion of synthetase-D to synthetase-I* and hence

accelerates glycogen synthesis while epinephrine and glucagons favours the

conversion of synthetase-I to synthetase-D, by their stimulating action of the

production of cyclic AMP, and hence inhibit glycogen synthesis. α β

5. Branching of glycogen. As soon as the straight chain polysaccharide attains a

length of eight glucose units, the enzyme amylo-1, 41, 6-trans-glucosidase

(branching enzyme) cleaves it into two fragments which then re-attach by

means of an α -1, 6-linkage.

(1, 4-Glucosyl unit)x (1, 4 Glucosyl and 1,6-glucosyl units)x

Glycogen

The existing branches of the glycogen molecule are extended by the

addition of new glucose molecules from UDPG under the influence of glycogen

158

synthetase. Thus under the combined action of glycogen synthetase and branching

enzyme, the glycogen molecule grows like a tree. The molecular weight of

glycogen thus synthesized may vary from 1 to 4 millions or more.

Inborn error of glycogen anabolism. A form of glycogen storage

deficiency resulting from inherited lack of glycogen synthetase has been

described. Such individuals are not being able to form proper amount of

glycogen. This inborn error of metabolism is characterized by fasting

hypoglycemia (less than normal blood glucose levels) with convulsions and

mental retardation. Normal individuals store approximately 100 gm. of glycogen

in the liver and about 250 gm. in muscles.

1.9.5 GLYCOGENOLYSIS.

The process of breakdown of glycogen to glucose (as in liver and kidney) or

glucose 6-phosphate (as in the muscles) is known as glycogenolysis. The process

involves the following steps

1. Cleavage of α -1, 4-linkage. The breakdown of glycogen is catalysed by the

enzyme phosphorylase in presence of inorganic phosphate. The reaction,

being known as phosphorolysis, splits up the terminal glucose as glucose 1-

phosphate. In this way a glycogen chain can be shortened by one glucose

unit at a time. It is important to note that the enzyme phosphorylase attacks

only the α -1, 4-linkages in glycogen, it can neither attack the α -1, 6-

linkages at the branching points nor can by-pass them to attack the next α -

1, 4-linkages. Thus if glycogen is subjected to the action of phosphorylase

alone, a shorter glycogen, known as limit dextrin is formed.

2. Cleavage of α -1, 6-linkages. The α -1, 6-linkages are cleaved by another

enzyme known as debranching enzyme (α -1, 6-glucosidase) which

removes the glucose unit linked by α -1, 6-linkage to free glucose and thus

allows the phosphorylase to continue its attack on the chain. Thus a

159

glycogen molecule can be completely broken down by the combined of

phosphorylase and debranching enzyme.

3. Conversion of glucose-1-phosphate to glucose-6-phosphate. Glucose-1-

phosphate obtained under the influence of phosphorylase is isomerised to

glucose 6-phosphate by

the action of

phosphoglucomutase.

4. Hydrolysis of glucose-

6-phosphate to glucose.

The glucose 6-

phosphate is then

hydroiysed under the

influence of glucose-6-phosphatase (present only in liver and kidney) to

free glucose. Thus glycogen is hydrolysed finally to free glucose in liver

and and kidney and to glucose 6-phosphate in muscles.

1.9.6 GLUCONEOGENESIS

A continuous supply

of glucose is necessary

as a source of energy

especially for the

nervous system and

the erythrocytes. For

example, man‘s brain

uses about 110 gm

glucose per day and

his blood converts about 24 gm. of glucose to lactate per day. On the basis the

minimum requirement of glucose in a normal adult man receiving no dietary

160

carbohydrate is more than 130 gm./day during rest and further increases during

exercise. In addition to this, glucose plays the following important roles.

i. It is required in adipose tissue as a source of glyceride-glycerol.

ii. It helps in maintaining the levels of intermediates of the citric cycle in

many tissues.

iii. Glucose is always required at a basal rate as a source of energy even under

conditions where fat supplies most of the caloric requirement of the

organism. Moreover, glucose is the only fuel that supplies energy to

skeletal muscles under anaerobic conditions.

iv. It is the precursor of lactose (milk sugar) in the mammary gland and it is

taken up actively by the fetus.

Thus it is not surprising to note that certain mechanisms have been

studied in certain specialized tissues for the conversion of non-carbohydrates

to glucose. The process of glucose (carbohydrate) synthesis from ono-

carbohydrate substances is known as gluconeogenesis (reversal of glycolysis)

and such non-carbohydrates substances are called as gluconeogenic

substances.

In mammals, this process occurs mostly in the liver and kidney which

show low glycolytic activity during gluconeogenesis. This process usually

occurs at a basal rate but becomes very active when diet is not able to meet the

carbohydrate requirement of the body at the required rate.

The most important gluconeogenic substances are lactic acid

(continuously produced in muscles and blood) and glycerol (from fats)

followed by propionic acid, certain amimo acids (derived from the dietary and

tissue proteins), certain α -keto acids such as pyruvic acid, α -ketoglutaric acid

and oxaloacetic acid. In general all the gluconeogenic susbstances at some

stage of their metabolism are linked with glycolytic or citric acid cycle

161

reactions and thus are converted into glucose of glycose or glycogen by the

reversal of these reactions.

The process of gluconeogenesis from lactate, amino-acids, propionic

acid and α -keto acids-takes place via the formation of oxaloacetic acid. The

whole of the process may be sketched as in Fig. 6.21, while the details of

glucose synthesis from the amino acids (the so called glucogenic or

antiketogenic amino acids) are discussed in the metabolism of individual

amino acids.

Physiological functions of gluconeogenesis : The process of

gluconeogenesis performs the folowing physiological functions.

i. During starvation (i.e. when aletary carbohydrates are not supplied) the

stored glycogen in the tissues of well-nourised man is exhausted after only

a few hours. At such critical times the tissues or dietary proteins break

down to yield glucogenic amino acids which may be converted into

glucose of glycogen and thus the process of gluconeogenesis helps in

maintaining the nomal blood sugar level at the times when dietary

carbohydrates are insufficient to meet the body carbohydrate requirement.

The process is especially important for nervous tissues which can derive

energy only from carbohydrates (not from fats1) and are irreparably

damaged if their supply of blucose is insufficient.

ii. The proces of gluconeogenesis brings about proper disposal of lactic acid

produced by the muscles during and after exercise and that of glycerol

produced in the adipose tissues.

iii. It also helps in establishing a dynamic equillibrium among carbohydrates,

fats and proteins and thus at the time of emergency one can be converted

into another.

162

2.0 LET US SUM UP.

Carbohydrates:

1. Carbohydrates are polyhydroxy aldehydes or ketones and their derivatives.

Their general formula is (CH2O)n. Originally were called as hydrates of

carbon. Monosaccharides are simple sugars e.g. glucose. It is the basic

building block of polysaccharides starch cellulose. Oligasaccharides have mo

to ten monosaccharides units joined together. Polysaccharides units joined

together in linear or branched chain. Eg. Cellulose

2. Carbohydrates

Monosaccharides

derivatives eg.

Glycosides

Deoxysugars;

Myoinositol,

Aminosugars,

N-acetyl muramic acid

Sialic acid.

Oligosaccharides Plysaccharides - Eg.

1Cellulose, Chitin-

structural Polysaccharides

2 Starch & Glycogen

Storage Polysaccharides.

3. Glycoproteins are a class of conjugated proteins which contain carbohydrate

group attacched covalently to the poly peptide chain. They are found in all

forms of life. They occur as cell-cbats, in blood, and in some proteins

harmones in vertabrates.

4. Glycolipids.

5. Carbohydrate metabolism

At the time of break down of carbohydrates, cell utilizes the enzymes of the

cytoplasm and mitochondria. The process involves a number of steps. The

163

steps that occur in cytoplasm comprises glycolysis, & kreb‘s cycle in

mitochondria

The Various steps in which oxidation of glucose into CO2 & H2O

1. Glycolysis or EMP pathway.

2. Kreb‘s cycle or citric acid cycle.

3. ETC

4. Oxidative phosphorylation

5. Biosynthesis of Ribose, a pentose sugar occurs through pentose phosphate

pathway.

6. The process of formation of D-glucose from non-carbohydrates precursors is

called gluconeogenesis. Important precursors are lactate, pyruvic acid,

glycerol, certain amino acids.

2.1 CHECK YOUR PROGRESS 1 THE KEY:

1. See sum up. Point 1.

2. A. occurs in DNA

3. Content of peptidoglycan.

4. In human tissue.

5. Structural component of plant cell.

6. Storage product of animal cell.

1. They are a group of related heteropolysaccharides containing two types of

alternating monosaccharides units with one acidic gr, either a carboxyl or

sulfuric gr.

CHECK YOUR PROGRESS 2: THE KEY:

1. It is a fermentative pathway that occurs in cytoplasm for glucose break down.

Net yield is – 8 ATP

164

2. a. Pyruvic acid

a. CO2 & H2O & utric acid is regenerated

b. Glycogen

2.2 ASSIGNMENT/ACTIVITY

1. Draw flow chart of Glycolysis, Kreb‘s Cycle, PPP, gluconeogenesis,

2. Write notes on structural and storage polysaccharides

3. List functions of carbohydrate

2.3 REFERENCES.

1. Agarwal‘s Textbook of Biochemistry (Physiological Chemistry) Goel

Publishing house meerut.

2. Lehninger, Biochemistry, Kalyani Publication.

3. Jain J.L., Biochemistry, S. Chand Publication.

4. Stryer Lubert, Biochemistry

5. Verma S.K. & Verma Mohit,

A text book of Plant Physiology, Biochemistry & Biotechnology, S.chand

Publication

165

UNIT-III LIPIDS

3.1 INTRODUCTION

3.2 OBJECTIVES

3.3 FATTY ACIDS

3.4 ESSENTIAL FATTY ACIDS

3.5 STRUCTURE AND FUNCTION OF TRIACYLGLYCEROLS

3.6 GLYCEROPHOSPHOLIPIDS

3.7 SPHINGOLIPIDS

3.8 CHOLESTEROL

3.9 BILE ACID

3.10 PROSTAGLANDINS

3.11 LIPOPROTEINS - COMPOSITION AND FUNCTION ROLE IN

ANTHEROSCLEROSIS

3.12 PROPERTIES OF LIPID AGGREGATES

3.12.1 MICELLES, BILAYERS, LIPOSOMES & THEIR POSIBBLE BILOGICAL

FUNCTIONS.

3.12.2 BIO-LOGICAL MEMBRANE

3.12.3 FLUID MOSAIC MODEL OF MEMBRANE STRUCTURE.

3.13 LIPID METABOLISM -OXIDATION OF FATTY ACIDS.

3.14 LET US SUM UP

3.15 CHECK YOUR PROGRESS: THE KEY

3.16 ASSIGNMENT / ACTIVITY

3.17 REFERENCES

3.1 INTRODUCTION

Lipids are esters of higher fatty acids. Lipids are water-insoluble organic

biomolecules that can be extracted from cells and tissues by nonpolar solvents, e.g.,

choloroform, ether, or benzene. There are several different families or classes of

lipids but all derive their distinctive properties from the hydrocarbon nature of a

major portion of their structure. (1) as structural components of membranes, (2) as

storage and trasport forms of metabolic fuel, (3) as a protective coating on the

surface of many organisms, and (4) as cell-surface components concerned in cell

recognition, species specificity, and tissue immunity. Some substances classified

among the lipids have intense biological activity; they include some of the vitamins

and hormones.

166

Although lipids are a distinct class of biomolecules, we shall see that they often

occur combined, either covalently or through weak bonds, with members of other

classes of bio-molecules to yield hybrid molecules such as glycolipids, lipoproteins,

which contain both lipids and proteins. In such biomolecules the distinctive chemical

and physical properties of their components are blended to fill specialized biological

functions.

3.2 OBJECTIVES

In this unit you are expected to

1. is removed Learn the general properties and

occurrence of lipids in cells.

2. There structure and possible biological functions.

3. Membrance Structure and metabolism of lipids.

3.3 FATTY ACIDS

Although fatty acids occur in very large amounts as

building block components of the saponifiable lipids, only

traces occur in free (unesterified) form in cells and tissues.

Over 100 different kinds of fatty acids have been isolated

from various lipids of animals, plants, and microorganisms.

All possess a long hydrocarbon chain and a terminal

carboxyl group. The hydrocarbon chain may be saturated,

as a in palmitic acid, or it may have one or more double

bonds, as in oleic acid; a few fatty acids contain triple bonds. Fatty acids differ from

each other primarily in chain length and in the number and position of their saturated

bonds. They are often symbolized by a shorthand notation that designates the length

167

of the carbon chain and the numbers, posistion, and configuration of the double

bonds. Thus palmitic acid (16 carbons, saturated) is symbolized 16:0 and oleic acid

[18 carbons and one double bond (cis) at carbons 9 and 10] is symbolized 18:19. it is

understood that the double bonds are cis unless indicated otherwise.

Some generalizations can be made on the different fatty acids of higher plants and

animals.

6. Even numbered straight chain fatty acids are found distributed both in plants

and animals, they are between 14 and 22 carbon atoms long, but those with 16

or 18 carbons predominate.In animal fats most abundantly found fatty acids

are palmitic acid (C16) and stearic acid (C18).

7. Unsaturated fatty acids predominate over the saturated ones, particularly in

higher plants and in animals living at low temperatures.

8. Unsaturated fatty acids have lower melting points than saturated fatty acids of

the same chain length

9. In most monounsaturated (monoenoic) fatty acids of higher organisms there is

a double bond between carbon atoms 9 and 10. In most polyunsaturated

(polyenoic) fatty acids one double bond is between carbon atoms9 and 10; the

additional double bonds usually occur between the 9, 10 double bond and the

methy-terminal end of the chain.

10. In most types of polyunsaturated fatty acids the double bonds are seperated by

one methylene group, for example, -CH=CH-CH2-CH=CH-; only in a few

types of plant fatty acids are the double bonds in conjugation, that is, -

CH=CH-CH=CH-.

3.4 ESSENTIAL FATTY ACID

168

When weanling or immature rats are placed on a fat-free diet, they grow poorly,

develop a scaly skin, lose hair, and ultimately die with many pathological signs.

When linoleic acid is present in the diet, these conditions do not develop. Linolenic

acid and arachidonic acid also prevent these symptoms. Saturated and

monounsaturated fatty acids are inactive. It has been concluded that mammals can

synthesize saturated and monounsaturated fatty acids from other precursors but are

unable to make linoleic and γ-linoleic acids. Fatty acids required in the diet of

mammals are called essential fatty acids. The most abundant essential fatty acid in

mammals is lionoleic acid, which makes up from 10 to 20 percent of the total fatty

acids of their triacylglycerols and phosphoglycerides. Linoleic and γ -linolenic acids

cannot be synthesized by mammals but must be obtained from plant sources, in

which they are very abundant. Linoleic acid is a necessary precursor in mammals for

the biosynthesis of arachidonic acid, which is not found in plants.

Although the specific functions of essential fatty acids in mammals were a mystery

for many years, one function has been discovered.

Essential fatty acids are necessary precursors in the

biosynthesis of a group of fatty acid derivatives called

prostaglandins, hormone like compounds which in trace

amounts have profound effects on a number of important

physiological activities.

3.5 STRUCTURE AND FUNCTION OF

TRIACYLGLYCEROLS (TRIGLYCERIDES)

Fatty acid esters of the alcohol glycerol are called

acylglycerols or glycerides; they are sometimes referred

169

to as "neutral fats," a term that has become archaic. When all three hydroxyl groups

of glycerol are esterified with fatty acids, the structure is called a triacylglycerol.

Triacyglycerols are the most abundant family of lipids and the major components of

depot or storage lipids in plant and animal cells. Triacyglycerols that are solid at

room temperature are often referred to as "fats" and those which are liquid as "oils"

Diacylglycerols (also called diglycerides) and monoacylglycerols (or

monoglycerides) are also found in nature, but in much samller amounts.

Triacylglycerols occur in many different types, according to the identity and position

of the three fatty acid components esterified to glycerol.

3. Those with a

single kind of fatty acid in all three positions, called simple triacylglycerols,

are named after the fatty acids they contain. Examples are tristearoylglycerol,

tripalmitoylglycerol, and trialeoyglycerol; the trivial and more commonly used

names are tristearin, tripalmitin, and triolein, respectively.

170

4. Mixed triacyglycerols contain two or more different fatty acids.

3.6 PHOSPHOGLYCERIDES

(GLYCEROPHOSPHOLIPIDS)

The second large class of complex lipids consists of the

phosphoglycerides, also called glycerol phosphatides. They

are characteristic major components of cell membranes; only

very small amounts of phosphoglycerides occur elsewhere in

cells. Phosphoglycerides are also loosely referred to as

phospholipids or phosphatides, but it should be noted that

not all phosphorus-containing lipids are phosphoglycerides;

e.g., sphingomyelin is a phospholipid because it contains

phosphorus, but it is better classified as a sphingolipid

because of the nature of the backbone structure to which the

fatty acid is attached.

In phosphoglycerides one of the primary hydroxyl groups of

glycerol is esterifed to phosphoric acid; the other hydroxyl

groups are esterfied to fatty acids. The parent compound of

the series is thus the phosphoric ester of glycerol. This

compound has an asymmetric carbon atom and can be

designated as either D-glycerol 1-phosphate or L-glycerol 3-

phosphate.

171

Phosphoglycerides possess a polar head in addition to their nonpolar hydrocarbon

tails, they are called amphiphatic or polar lipids.The different types of

phosphoglycerides differ in the size, shape, and electric charge of their polar head

groups. Each type of phosphoglyceride can exist in many different chemical species

differing in their fatty acid substituents. Usually there is one saturated and one

unsaturated fatty acid, the latter in the 2 position of glycerol.

The parent compound of the phosphoglycerides is phosphatidic acid, which contains

no polar alcohol head group. It occurs is only very small amounts in cells, but it is an

importnat intermediate in the biosynthesis of the phosphoglycerides. The most

abundant phosphoglycerides in higher plants and animals are

phosphatidylethanolamine and phosphatidylcholine, which contain as head groups

the amino alcohols ethanolamine and choline, respectively.

Plasmologens differ from all the other phosphoglycerides.

3.7 SPHINGOLIPIDS

Sphingolipids, complex lipids containing as their backbone sphingosine or a related

base, are important membrane compoenents in both plant and animal cells. They are

present in especially large amounts in brain and nerve tissue. Only trace amounts of

sphingolipids are found in depot fats. All sphingolipids contain three characteristic

building-block components: one molecule of a fatty acid, one molecule of

sphingosine or one of its derivatives, and a polar head group, which in some

sphingolipids is very large and complex.

Sphingosine is one of 30 or more different long-chain amino alcohols found in

sphingolipids of various species. In mammals sphingosin and dihydrosphingosine are

the major bases of sphingolipids, in higher plants and yeast phytosphingosine is the

major base, and in marine invertebrates doubly unsaturated bases such as 4, 8-

sphingadiene are common. The sphingosine base is connected at its amino group by

an amide linkage to a long saturated or monounsaturated fatty acid of 18 to 26

172

carbond atoms. the resulting compound, which has two nonpolar tails and is called a

ceramide, is the characteristic parent structure of all sphingolipids. Different polar

head groups are attached to the hydroxyl group at the 1 position of the sphingosine

base.

Sphingomyeline

The most abundant sphingolipids in the tissues of higher animals

are

sphingomyelins, which contain phosphorylethanolamine or phosphorylcholine as

their polar head groups, esterifed to the 1-hydroxyl group of ceramide.

Sphingomyelines have physical properties very similar to those of

phosphatidylethanolamine and phosphatidylcholine; they are zwitterions at pH 7.0.

Neutral Glycosphingolipids

A second class of sphingolipids contains one or more neutral sugar residues as their

polar head groups and thus has no electric charge; they are called neutral

glycosphingolipids. The simplest of these are the cerebrosides, which contain as their

polar head group a monosaccharide bound in β -glycosidic linkage to the hydroxyl

group of ceramid. The cerebrosides of the brain and nervous system contain D-

galactose and are therefore called galactocerabrosides.

Neutral glycosphingolipids with disaccharides as their polar head groups are called

dihexosides. Also known as trihexosides and tetra hexosides.

173

Acidic Glycosphingolipids (Gangliosides)

The third and most complex group of glycosphingolipids are the gangliosides; they

contain in their oligosaccharide head groups one or more residues of a sialic acid,

which gives the polar head of the gangliosides a net negative charge at pH 7.0

Waxes

Waxes are water-insoluble, solid esters of higher fatty acids with long-chain

monohydroxylic fatty alcohols or with sterols. They are soft and pliable when warm

but hard when cold. Waxes are fond as protective coatings on skin, fur, and feathers,

on leaves and fruits of higher plants, and on the exoskeleton of many insects. The

major components of beeswax are palmitic acid esters of long-chain fatty alcohols

with 26 to 34 carbon atoms. Lanolin, or wool fat, is a mixture of fatty acid esters of

the sterols lonosterol and agnosterol.

Simple (Nonsaponifiable) Lipids

The lipids discussed up to this point contain fatty acids are building blocks, which

can be released on alkaline hydrolysis. The simple lipids contain no fatty acids. They

occur in smaller amounts in cells and tissues than the complex lipids, but they

include many substance having profound biological activity- vitamins, hormones,

and other highly specialized fat-soluble

biomolecules.

3.8 STEROIDS (CHOLESTEROL)

Steroids are derivatives of the saturated

tetracylic hydrocarbon perhydrocyl-

opentanophenanthrene. A great many

different steroids, each with a distinctive

174

function of activity, have been isolated from natural sources. Steroids differ in the

number and position of double bonds, in the type, location, and number of

substituent functinal groups, in the configuration (α or β) of the bonds between the

substituent groups and the nucleus, and in the configuration of the rings in relation to

each other, since the parent hydrocarbon has six centers of asymmetry. The main

points of substitution are carbon 3 of ring A, carbon 11 of ring C, and carbon 17 of

ring D. All steroids originate from the linear triterpene squalene, which cyclizes

readily. The first important steroid product of this cyclization is lanosterol, which in

animal tissues is the precursor of cholestrol, the most abundant steroid in animal

tissues. Cholesterol and lanosterol are members of a large subgroup of steroieds

called the sterols. Cholestrol melts at 1500 C and is insoluble in water but readily

extracted from tissues with chloroform, ether, benzene, or hot alcohol. Cholestrol

occurs in the plasma membranes of many animal cells and in the lipoproteins of

blood plasma. Cholestrol occurs only rarely in higher plants, which contain other

types of sterols known collectively as phytosterols. Among these are stigmasterol

and sitosterol. Fungi and yeasts contain still other types of sterols, the mycosterols.

Cholesterol is the precursor of many other steroids in animal tissues, including the

bile acids, detergentlike compounds that aid in amulsification and absorption of

lipids in the intestine; the androgens, or male sex hormones; the estrogens, or female

sex hormones; the progestational hormone progesterone; and the adrenocortical

hormones.

175

3.9 BILE ACIDS

These are also

steroid

compounds and are formed from cholesterol in the body.In the bile of higher animals

,cholic deoxycholic and lithocholic acids are found.They are conjugated with glycinr

and taurine forming peptide bonds at the COOH group,resulting glychocolic and

taurocholic acids.They may be considered as derivatives of cholanic acid.

3.0 PROSTALGLANDINS

Prostaglandins are a family of fatty acid derivatives which have a variety of potent

biological activities of a hormonal or regulatory nature. The name prostaglandin was

first given in the 1930s by the Swedish physiologist U.S. von Euler to a lipid-soluble

176

acidic substance found in the seminal plasma, the prostate gland, and the seminal

vesicles. At least 14 prostaglandins occur in human seminal plasma, and many others

have been found in other tissues or prepared synthetically in the laboratory.

The structure of prostaglandins was established by S. Bergstrom and his colleagues

in Sweeden. All the natural prostaglandins are biologically derived by cyclization of

20 carbon unsaturated fatty acids, such as arachidonic acid, which is formed from the

essential fatty acid linoleic acid. Five of the carbon atoms of the fatty acid back-bone

are looped to form a five-membered ring. The prostaglandins are named according to

their ring substituents and the number of additional side-chain double bonds, which

have the cis configuration. The best known are prostaglandins E1, F1α, and F2α,

abbreviated as PGE1, PGF1α and PGF2α, respectively. These in turn are the parent

compounds of further biologically active prostaglandins.

The prostaglandins differ from each other with respect to their bilogical activity,

although all show at least some activity in lowering blood pressure and inducing

smooth muscle to contract. Some, like PGE1, antagonize the action of certain

hormones. PGE2 and PGE2α may find clinical use in inducing labor and bringing

about therapeutic abortion.

CHECK YOUR PROGRESS – 1

Note: Write your answer in the space given below.

Check your answer with the one at the end of the unit.

Fill in the blanks.

10. Lipids are ________ of higher fatty acids.

11. Fluid mosaic model of membrane was proposed by ________

12. Glycerol phosphatides is another name of ________.

13. ________ is most abundant in the tissues of higher animals.

14. Steroids are ________.

177

3.1 LIPOPROTEIN COMPOSITION AND FUNCTION, ROLE IN

ANTHEROSCLEROSIS

Certain lipids associate with specific proteins to form lipoprotein systems in which

the specific physical properties of these two classes of biomolecules are blended.

There are two major types, transport lipoproteins and membrane systems. In these

systems the lipids and proteins are not covalently joined but are held together largely

by hydrophobic interactions between the nonpolar portions of the lipid and the

protein components.

Antherosclerosis (Greek – adhere-mush) is a complex disesase charaterized by

hardening of artries due to accumlation of lipids.

Hyper cholesterolemia is associated with anthero scholerosis and coronary heart

disease. Antherosclerosis is characterized by deposition of cholesteryl esters and

other lipids in the intima of the arterial walls often leading to hardening of coronary

artries and arteral blood vessels LDL – cholesterol is positively related, where as

HDL-cholesterol is negatively co-related with cardio vascular diseases (LDL stands

for lethaly dangerous lipoprotein & HDL is highly desirable lipoprotein)

CAUSE OF ANTHERROSCLEROSIS & CHD :-

Antherosis deve & CHD related to plasma cholesterol & LDL Plasma HDL is

inversly related to LHD.

DISORDERS THAT MAY CAUSE ANTHEROSCLEROSIS:

Certain diseases are associated with it like dieabetes mellitus, hyper lipoproteins,

nephrotic syndrome, hypthyroidism etc. obesity, smoking, high consumption of fat,

lack of physical exercise, stress etc. are probable cause of anthersclerosis.

178

Antioxidants in general decreases the oxidation of LDL studies suggest that taking of

antioxidants reduces the risk of antherosclerosis.

CONTROL OF HYPER CHOLESTEREMIA

Various measures to lower plasma cholesterol are –

1- Consumption of PUFA :- dietary entake of polyunsaturated F.A. reduces

cholesterol level. PUFA rich oils are soyabean oil. Cornoil, fish oil –etc

2- Dietary cholesterol :- avoidance of cholesterol rich food i.e. animal food is

recommended.

3- Dietary fibers : - fibers present in vegetable decreases cholesterol

absorption from intestine.

4- Avoidance of high carbohydrate diet:- diets rich in carbohydrates if

avoided controls hyper cholestrolemia.

5- Use of drugs :- Drugs such as lovastatin which inhibit HMG CoA reductase

& decrease cholesterol sym.

Transport Lipoproteins of Blood Plasma

The plasma lipoproteins are complexes in which the lipids and proteins occur in a

relatively fixed ratio. They carry water-insoluble lipids between various organs via

the blood, in a form with a relatively samll and constant particle diameter and

weight. Human plasma lipoproteins occur in four major classes that differ in density

as well as particle size.

3.2 PROPERTIES OF LIPID AGGREGATES

3.2.1 LIPID MICELLES AND BILAYERS

When a polar lipid, like a

phosphoglyceride, is added to water,

only a small fraction dissolves to form

a true molecular solution. Above the

179

critical micelle concentration the polar lipids associate into various types of

aggregates resembling the micelles formed from soaps. In such structures the

hydrocarbon tails are hidden from the aqueous environment and form an internal

hydrophobic phase whereas the hydrophilic heads are exposed on the surface.

Triacylglycerols do not form such aggregates since they have no polar

heads.Phosphoglycerides also form monolayers on air-water interfaces as well as

bilayers separating two aqueous compartments. Liposomes are completely closed,

vasicular bilayer structures formed by exposing phosphoglyceride-water suspensions

to sonic oscillation. Bilayer systems of this sort have been extensively studied as

models of natural membranes, which appear to contain polar phospholipid bilayers

as their continuous phase.

3.2.2 BIOLOGICAL MEMBRANE STRUCTURE

The

most

180

satisfactory model of membrane structure to date appears to be the fluid-mosaic

model, postulated by S.J. Sanger and G.L. Nicolson in 1972. This model postulated

that the phospholipids of membranes are arranged in a bilayer to form a fluid, liquid-

crystalline matrix. The fluid-mosaic model postulates that the membrane proteins are

globular. Some of the proteins are partially embedded in the membrane, penetrating

into the lipid phase from either side, and others completely span the membrane

.It views membranes as a fluid mosaic in which proteins are inserted into a lipid

bilayer. While phospholipids provide the basic structural organization of membranes,

membrane proteins carry out the specific functions of the different membranes of the

cell. These proteins are divided into two general classes, based on the nature of their

association with the membrane. Integral membrane proteins are embedded directly

within the lipid bilayer. Peripheral membrane proteins are not inserted into the lipid

bilayer but are associated with the membrane indirectly, generally by interactions

with integral membrane proteins.The fluid-mosaic model accounts satisfactorily for

many features and properties of bilogical membranes.

1. It provides for membranes with widely different protein content, depending on

the number of different protein molecules per unit area of membrane.

2. It provides for the varying thickness of different types of membranes.

3. It can account for the asymmetry of natural memebranes, since it permits

proteins of different types to be arranged on the two surfaces of the lipid

bilayer.

4. It accounts for the electrical properties and permeability of membranes.

5. It also accounts for the observation that some protein components of cell

membranes move in the plane of the membrane at a rather high rate.

181

a. LIPID METABOLISM

b. As you know metabolism involves both anabolism and catabolism, here we will

learn anabolism/biosynthesis of fatty acid first and then their breakdown.

FATTY ACID BIOSYNTHESIS

Since, two types of fatty acids Viz , saturated and unsaturated fatty acid occurs in

nature different pathways are followed for synthesis of different F.A

1.Biosynthesis of saturated F.A. (malonyl CoA pathway)

2.Enzymatic biosynthesis of unsaturated F.A.

(i) Biosynthesis of saturated Fatty Acids: Naturally occurring fatty acids may be

saturated and main pathway of biosynthesis of fatty acid in plants, animals and

bacteria is common and takes through malonyl CoA pathway. In fatty

acids(participating in fat synthesis) the number of carbon atoms varies form 16 to

18.Complete biosynthesis of fatty acid takes place in cytosol. Overall reaction is

catalysed by the complex of 7 proteins-the fatty acid synthetase complex. Ultimate

source of carbon atoms of fatty acid is acetyl-CoA which is produced from

carbohydrates and aminoacids. Acetyl-CoA is regenerated with the help of citrate

cleaving enzymes as follows:

Citrate Cleaving

Citric Acid + CoA +ATP Acetyl-CoA + ADP+Pi +

OAA

Enzymes

182

Acetyl-CoA acts as a primer.

Molecules of malonyl-CoA are successively attached to the primer

molecules of Acetyl-CoA accompanied by decarboxylation.

Before starting of fatty acid biosynthesis an important preparatory

reaction, the formation of malonyl-CoA takes place in cytosol.

According to Green (1960). Two enzymes complexes and five

cofactors-ATP, Mn++

, biotin, NADPH and CO2 are essential for the

synthesis of fatty acids.

The long chain compounds of fatty acids are synthesized from two

carbon compounds Acetyl-coenzyme A (Acetyl-CoA) which is highly

reactive compound and is produced as an intermediate in respiration of

sugar and fats.

The synthesis of fats takes place in stepwise reaction taking place again

and again.

In each step, 2-carbons atoms of acetyl-CoA are added in the chain.

In presence of biotin-acetyl-CoA carboxylase enzyme, acetyl-CoA react

with CO2 and ATP to produce malonyl-CoA,ADP and inorganic

phosphate. Mn++

acts as cofactor in this reaction.

183

Carboxylase

CH3 – CO – SCOA+CO2+ATP (Acetyl – CoA)(2C)

Biotin – Acetyl - CoA Mn++

H O

| ||

H__

C__

C __

S.CoA + ADP +IP

|

O == C__

OH

Malonyl-CoA(3C)

Malony I-CoA react with acetyl-CoA to produce acetomalonyI-CoA (5C

compound).

H O

| ||

H__

C __

C __

S.CoA+CH3___

CO___

S.CoA

|

O==C__

OH

Malonyl-CoA(3C) Acetyl-Coa(2C)3

H O H O

| || | ||

H__

C__

C __

C __

C__

S.CoA

| |

H O = C_ OH

Acetomalonyl-CoA(5C)

In presence of specific enzyme and coenzyme NADPH, acetomalonyl-CoA is

con verted into 4 carbon compound butyryl-CoA. CO2 is released during this

reaction and water (H2O) and NADP are formed.

H O H O

| || | ||

H__

C __

C __

C __

C __

S.CoA+ 4NADPH

| |

H O == C__

OH

Acetomalony-CoA(5C)

H H H O

| || | ||

H__

C __

C __

C __

C __

S.CoA+CO2 +4NADP=H2O

| | |

184

H H H

Butyryl-cOa(4C)

The butyryl-CoA then reacts with another molecule of malonyl-CoA and cycle

is repeated and 6-carbon compounds is produced. This will again react with

still another molecule of malonyl-CoA to produce 8-carbon compounds. When

the chain length reaches 16 or 18-carbons, the fatty acid is released.

(ii) Biosynthesis of unsaturated fatty acid: Biosynthesis of unsaturated fatty acid has

not been completely studied in higher plants. During their synthesis, double bonds

are introduced into previously formed saturated fatty acid by the enzyme desaturase.

Enzymes acyl-CoA desaturase and stearyl-CoA desaturase have been isolated from

yeast and Euglena respectively.

OXIDATION OF LIPIDS OR DEGRADATION OF FATS.

Lipid is a storage material and can be used as energy rich fuel by cells during seed

germination. Degradation or oxidation of fats involves following three processes:

1) Hydrolysis of fat into glycerol and fatty acids

2) Metabolism of glycerol

3) Oxidation of fatty acids.

I) Hydrolysis of Fat into Glycerol and Fatty Acids

Degradation of fatty acids starts with their hydrolysis. During seed germination, the

enzyme lipase catalyses this reaction. During this process triglycerides react with

water to produce fatty acids and glycerol. The whole process is completed in three

steps.The fats first split to produce diglycerides,part of these are then split to

monoglycerides.Finally part of the monoglycerides split to yield fattyacid and

glycerol.This reaction occurs at alkaline pH.

2) Metabolism of Glycerol :

185

Glycerol is produced during hydrolysis of triglycerides, enters the

carbohydrates metabolism and metabolized into CO2 and H2O through various steps.

According to Stumpf (1955) and Beevers (1956), the metabolism of glycerol takes

place according to following diagram(Figure 13.9). After complete metabolism,

glycerol is converted into acetyl-CoA, which may be oxidized into Krebs cycle to

CO2 and H2O .

3) Oxidation of Fatty Acids:

The oxidation of fats depend upon α- or β-carbon atom. On the basis of α- and

β-carbon atom, the oxidation of fatty acids is of the following kinds:

(i) α- oxidation , (ii) β- oxidation.

α- OXIDATION OF FATTY ACIDS

α- oxidation of fatty acids is discovered by Newcomb and stumpf in 1952. this

type of oxidation is found only in the fats in which number of carbon atoms is

limited from 13 to 18 carbon. In this oxidation only one carbon atom in every step. It

is called α-oxidation because in this process oxidation of α-carbon atom takes place.

* α-oxidation of fatty acids takes place only in cotyledons and young leaves.

Mechanism of α-oxidation

The α-oxidation is completed in following two steps:

a. Decarboxylation: First of all in the presence of fatty acid peroxidase

enzyme, peroxidative decarboxylation of fatty acids takes place. In this

process fatty acids react with hydrogen peroxide(H2O2) to yield an

aldehyde (one carbon atom shorter than fatty acid) CO2 and H2O.

Peroxidase

R-CH2- CH2-COOH + H2O2 R-CH2-CHO + CO2 +

H2O

α-carbon fatty acids Hydrogen Peroxide Aldehyde

186

b. Dehydrogenation : the enzyme aldehyde dehydrogenase now catalyses

the oxidation of aldehyde(formed by first enzyme)to yield the

corresponding acid.

Dehydrogenase

R-CH2-CHO + H2O R-CH2-COOH

Aldehyde Acid

NAD+

NADH + H+

This resulted acid is again utilized by the enzyme fatty acid peroxidase as substrate

for another turn round the two stage of α-oxidation spiral. The enzyme of α-

oxidation are specific for long chain saturated fatty acids. The acids with more than

C12(usually C14,C16,and C18)are utilized as substrate in α-oxidation.

*The fatty acids formed after dehydrogenation may also be oxidized through β-

oxidation.

β-OXIDATION OF FATTY ACID

According to Knoop, degradation of fatty acids takes place by successive removal of

C2 units after oxidation of the β-carbon atoms. β-oxidation is the chief process of

fatty acids degradation in plants. β-oxidation takes place in mitochondrial matrix

(and also in glyoxyzones) and involves sequential removal of 2-C in the form of

acetyl-CoA molecules from the carboxyl and of fatty acids. This is called β-oxidation

because β-carbon (i.e.C-3) of the fatty acid is oxidized during this process.

Requirements of β-oxidation

β-oxidation requires the following substance:

a) A fatty acid

187

b) An energy source- ATP

c) Coenzyme-A

d) A carrier molecule – carnitine

e) five enzymes:

1) Acetyl-CoA synthetase

2) Acetyl-CoA dehydrogenase

3) Enoyl-CoA hydrase

4) β-hyrdoxy acyl CoA dehydrogenase

5) Thiolase

Mechanism of β-oxidation :

Fatty acids are formed in cytosol. Now the question is, how fatty acids enter

mitochondria for their degradation:

These are the following steps by which fatty acid enters mitochondria:

a. Activation and entry of fatty acid into mitochondria

i. Activation of fatty acids into mitochondria.

ii. Transfer to carnitine.

iii. Transfer to intramitochondrial membrane.

188

b. OXIDATION OR DEGRADATION OF FATTY ACID

1) Activation and entry of fatty acids into mitochondria: It is completed in following

three steps:

(a) Activation of fatty acids: the first step involves the activation of fatty

acid in the presence of ATP and enzyme thiokinase. CoASH is consumed and

CoA derivative of fatty acid is produced. In this reaction esterification of fatty

acid takes place.

Thiokinase,Mn++

R.CH2.CH2COOH+CoASH+ATP ==============

189

RCH2CH2C.SCoA

|| + AMP+PPi

O

Fatty acyl-CoA Pyrophosphate

(b) transfer to carnitine: the acyl group of fatty acid CoA is transferred to

carnitine.

Carnitine s a carrier protein which is found in inner mitochondrial

membrane which transport fatty acyl CoA through inner mitochondrial membrane to

the actual site of degradation.

CH3

R__

CH2.CH2 C O__

CH__

CH2__

N+ CH3

|| | CH3

O CH2COO_

Fatty acyl-CoA Carnitine

Acyl-CoAcarnitine transferase

CH3

R__

CH2.CH2 C__

O__

CH__

CH2__

N+ CH3 + COASH

|| | CH3

O CH2COO_

Fatty acyl-CoA Carnitine

(C) Transfer to intramitochondrial membrane: Dergadation of fatty acid takes place

in mitochondrial matrix, which requires Acyl-CoA as substrate. In this step acyl

group of fatty acyl carnitine is transferred to intramitochondrial CoaA.

SCoA+H

190

CH3

R__

CH2.CH2 C__

O__

CH__

CH2__

N+ CH3 + COASH

|| | CH3

O CH2COO_

Fatty acyl-CoA Carnitine

Acyl-CoAcarnitine transferase

CH3

R__

CH2.CH2 C__

S__

CoA+OH__

CH__

CH2__

N+

CH3

|| | CH3

O CH2__

COO_

Fatty acyl S.CoA Carnitine

2) Oxidation or degradation of fatty acid : The oxidation of fatty acid involves in

the following fur steps:

(a) First dehydrogenation : During oxidation, first of all two hydrogen

atoms are reomoved from α- and β-carbon atoms of fatty acyl-CoA and

trans – α, β-unsaturated fatty acyl-CoA is formed. This reaction is

catalysed by FAD containing enzyme acyl-CoA dehydrogenase.

β α Acyl CoA dehydrogenase

R – CH2-CH2-CO-S-CoA + FAD

191

β α

R-CH=CH-CO-S-CoA+

FADH2

Trans α,β-unsaturated fatty acid

b) Hydration: In the second step the addition of water molecule takes

place to form ,β-hydroxyacyl-CoA in the presence of Enoyl hydrase.

Enoyl Hydrase

R-CH=CH-CO-S-CoA +HOH

R-CH(OH)-CH2-CO-S-CoA

β -Hydroxy fatty acyl- CoA

c) Second dehydrogenation: Now β-hydroxy acyl-CoA is

dehydrogenated in the presence of NAD specific β- hydorxy acyl-CoA

dehydrogenase. Two hydrogen atoms are removed from β-C atom (β-

oxidation), which now bears a carboxyl function and β-keto fatty acyl-

CoA is formed.

β- hydorxy acyl-CoA

R-CH(OH)-CH2-CO-S-CoA + NAD+

Dehydrogenase

R-CO-CH2-CO-S-CoA + NADH

+H+

β-keto fatty acyl-CoA

192

d) Thiolysis : β- fatty acyl-CoA is unstable and it releases 2-C fragment

as actyl –CoA by the process of thiolysis. The thioclastic cleavage of β-

keto fatty acyl-CoA takes place in the presence of enzyme β-keto acyl-

thiolase and results in the formation of an active 2-C unit actyl-CoA and

β-keto fatty acyl-CoA molecule which is shorter by 2-C atoms then

when it entered the β-oxidation spiral.

*Acetyl CoA is used in TCA (=Krebs cycle) and Keto acyl-CoA

reenters into β-oxidation for its complete oxidation and in every step

two carbon atoms are released. The sequence continues until whole

molecule is degraded.

e.g.palmitic acid enters seven times in the β-oxidation pathway for their

complete oxidation.

Energetics of β-oxidation

For complete oxidation of palmitic acid, it is passed through β-oxidation

pathway seven times and get completely oxidized to form CO2 and H2O.

C16H32O2 + 23O2 16CO2 + 16H2O

Palmitic acid

*Each turn of β-oxidation pathway produces 5ATP molecules, however the first

turn shows a net gain of only 4 ATP, one ATP molecule is utilized in activating the

fatty acid molecule.

* Each acetyl CoA molecule after complete oxidation through TCA cycle

produces 12 ATP molecules. Thus , the total number of ATP molecules produced by

a fatty acid depends upon the number of carbon atoms present in that fatty acid

molecule.

For e.g.: one molecule of palmitic acid (C16) after complete oxidation to CO2 and

H2O produces 130 molecules of ATP as follows:

193

1. During activation 1 ATP is udes and 8

energy rich acetyl-CoA are formed.

2. FADH2 and 1 NADH2 are formed in

each cycle. Their reoxidation takes place through ETS

Step 1 :

Palmitic acid 8 actyl –CoA +14 electron pairs

7 pairs of electrons via FAD+

7*2 = 14 ATP

7 pairs of electrons via NAD+

7*3 = 21 ATP

35 ATP

1 ATP used in activation of fatty acid – 1 ATP

Net 34 ATP

Step 2 : Now Acetyl-CoA enters the T.C.A cycle and oxidized. As we

know that 3 ATP molecules are formed from each O2 atom during oxidative

phosphorylation. Here 32 atoms of O2 are used, hence,

T.C.A. cycle

8 Acetyl-CoA + 16 O2 16 CO2 + 8H2O + 8CoA

Therfore 32*3 = 96 ATP formed.

Thus total ATP formed will be 34 + 96 = 130 ATP molecules gained. The efficiency

can be calculated

130*8000*100

= = 49%

2340,000

The remaining energy is lost in the form of heat.

194

195

CHECK YOUR PROGRESS – 2

3.4 LET US SUM UP.

Lipids are esters of higher fatty acids. There are several different families or

classes of lipids but all derive their distinctive properties from the hydrocarbon

nature of a major portion of their structure

Lipids are the important constitutnets of cell membranes. Fluid mosaic model

of the membrane is the most accepted model of membrane structure.

3.5 CHECK YOUR PROGRESS 1 : THE KEY

1. Esters

2. Sanger and Nicholson in 1972

3. Phosphoglycerides

4. Sphingomylines

Note: Write your answer in the space given below.

Compare your answer with the one given at the end of the unit.

Q.1 Write Short notes on.

a. Fluid mosaic model of membrane.

b. -oxidation of F-A

Q.2 What are liposomes. --------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

196

5. Derivatives of the Saturated tetracyclic hydrocarbon perhydrocyclopentano

phenanthrene.

CHECK YOUR PROGRESS 2 : THE KEY

Hint

Q.a 1 Draw diagram of fluid mosaic model of membrane.

2 Write about structure and function of protein molecules

3 Explain structue of phospholipids.

b Draw pathway & mention names of enzymes also.

2 Liposomes are membrane bound molecules used for target directed drug

delivery.

3.6 ASSIGNMENT/ACTIVITY

1. Prepare a model of cell membrane.

2. Draw a chart showing lipid classification.

or

3. Draw a chart showing

β-oxidation of lipids.

3.7 REFERENCES.

6. Agarwal‘s Textbook of Biochemistry (Physiological Chemistry) Goel

Publishing house meerut.

7. Lehninger, Biochemistry, Kalyani Publication.

8. Jain J.L., Biochemistry, S. Chand Publication.

9. Stryer Lubert, Biochemistry

10. Verma S.K. & Verma Mohit, a Text Book of Plant Physiology, Biochemistry

& Biotechnology, S. Chand Publication

197

UNIVERSITY - MADHYA PRADESH BHOJ OPEN UNIVERSITY

BHOPAL (M.P.)

PROGRAMME - M.Sc.P.chemistry

PAPER - v (A-II)

TITLE OF PAPER - BIOLOGY FOR CHEMIST

BKOCK NO . - III

UNIT WRITER - UNIT - I Smt. Shikha Mandloi

Asst. Prof. Microbiology

Sri Sathya Sai College for Women

UNIT – II Smt. Shikha Mandloi

Asst. Prof. Microbiology

Sri Sathya Sai College for Women

EDITOR - Dr.(Smt.) Renu Mishra, HOD, Botany &

Microbiology,

Sri Sathya Sai College for Women, Bhopal

COORDINATION

COMMITTEE - Dr. Abha Swarup, Director, Printing & Translation

Major Pradeep Khare, Consultant, Printing &

Translation

198

POST GRADUATE PROGRAMME

M.Sc.P. CHEMISTRY

DISTANCE EDUCATION

SELF INSTRUCTIONAL

MATERIAL

Paper-v(A-II)

BIOLOGY FOR CHEMISTS

MADHYA PRADESH BHOJ OPEN UNIVERSITY

BHOPAL (M.P.)

199

INTRODUCTION

Proteins are the most abundant molecules in cells, consisting 50 percent or more of their dry weight. They are found in every part of every cell, since they are fundamental in all aspects of cell structure and function. There are many different kinds of proteins, each specialized for a different biological function. Moreover, most of the genetic information is expressed by proteins. Proteins consist of long chains, in which amino acids occur in specific linear sequences. Yet we know that in each type of protein the polypeptide chain is folded into a specific three-dimensional conformation, which is required for its specific biological function and activity. In this unit, we examine various aspects of the primary structure of proteins, which we have defined as the covalent backbone structure of polypeptide chains, including the sequence of amino acid residues. The three major aspects covered are: (1) the determination of amino acid sequence in polypeptide chains, (2) the significance of variations in the amino acid sequences of different proteins in different species, and (3) the laboratory synthesis of polypeptide chains.(4) structure of proteins The nucleic acids are of considerable importance in biological systems.Two types of nucleic acids are found in the cells of all living organisms. These are: 1. Deoxyribonucleic acid - DNA 2. Ribonucleic acid - RNA The name nucleic acid was given to it after knowing its acidic property. They are of two types; (1) Ribose nucleic acid, and(2) Deoxyribose nucleic acid . The basic chemical subunits of the nucleic acids are nucleotides. The nucleotides are made up of three components: (i) A heterocyclic ring containing nitrogen, known as a nitrogenous base, (ii) a five carbon pentose sugar, and (iii) A phosphate group. The bases found in nucleic acid are of two kinds- purines and pyrimidines.Adenine and guanine are purine and cytosine, uracil and thymine are pyrimidine bases. The nucleotides found in

200

nucleic acids are much fewer in number than the α-amino acids. DNA is found in almost all the cells as a major component of chromosomes of the nucleus. Certain viruses, including many of the bacterial viruses or bacteriophages, are DNA-protein particles. Mostly the plant viruses are RNA-protein particles. Ribose nucleic acid (RNA) is also of common occurrence in plants as well as animals. It is of three types- (i)ribosomal RNA (r-RNA); (ii) soluble RNA or transfer RNA (t-RNA) and (iii) messenger RNA (m-RNA). Ribosomal-RNA is found in small sub-cellular particles, the ribosomes. RNAs with sendimentation Coefficient value, 5S, 16S and 23S have been reported from 70S ribosomes, while 18S, 28S, 5.8S and 5S r-RNAs have been reported from 80S ribosomes. T-RNA is found in free from in the cytoplasm. M-RNA is found in small quantities in association with ribosomes.

Unit IV

AMINO ACIDS, PEPTIDES & PROTEINS

1.1 Introduction

1.2 Objectives

1.3 Chemical & enzymatic hydrolysis of proteins to peptides &

amino acid

sequencing

1.4 Structure of proteins

1.4:1 Forces responsible for holding secondary structure

1.4:2 α-helix, β-sheets, super-secondary structure

1.4:3 Structure of collagen

1.4:4 Tertiary structure- folding & domain structure

1.4:5 Quaternary structure

1.5 Amino acid metabolism degradation & Biosynthesis of amino

acid

sequence determination

201

1.6 Chemistry of oxytocin & tryptophan releasing hormone.

1.7 Let us sum up

1.8 Check your progress – The Key

1.9 Assignment / Activity

2.0 References

1.1 INTRODUCTION

Proteins are the most abundant molecules in cells, consisting 50 percent or

more of their dry weight. They are found in every part of every cell, since they

are fundamental in all aspects of cell structure and function. There are many

different kinds of proteins, each specialized for a different biological function.

Moreover, most of the genetic information is expressed by proteins. The

structure of protein molecules and its relationship to their biological function

and activity are central problems in biochemistry today.

Proteins consist of long chains, in which amino acids occur in specific linear

sequences. Yet we know that in each type of protein the polypeptide chain is

folded into a specific three-dimensional conformation, which is required for its

specific biological function and activity. How is the linear, or one-dimensional,

information inherent in the amino acid sequence of polypeptide chains

202

translated into the three-dimensional conformation of native protein

molecules?

The answer to this question comes from some of the most significant

advances in modern biological research. These discoveries, made possible

by the application of physical-chemical measurements to pure proteins, have

illuminated the function and comparative biology of proteins.

In this chapter, we examine various aspects of the primary structure of

proteins, which we have defined as the covalent backbone structure of

polypeptide chains, including the sequence of amino acid residues. We begin

by considering the properties of simple peptides. Then we examine three

major aspects: (1) the determination of amino acid sequence in polypeptide

chains, (2) the significance of variations in the amino acid sequences of

different proteins in different species, and (3) the laboratory synthesis of

polypeptide chains.(4) structure of proteins

1.2 OBJECTIVES :-

This unit emphasizes on developing a

1. Basic understanding of the structure of amino acids and proteins.

2. Make you understand the various aspects and forces involved in formation

of primary, secondary tertiary and quarternary structure of proteins.

3. Information about general occurrence and distribution of proteins in living

system.

203

1.3 CHEMICAL & ENZYMATIC HYDROLYSIS OF PROTEINS INTO

PEPTIDES & AMINO ACID SEQUENCY

The structure of peptide

Simple peptides containing two, three, four, or more amino acid residues,

i.e., dipeptides, tripeptides, tetrapeptides, etc., joined covalently through

peptide bonds, are formed on partial hydrolysis of much longer polypeptide

chains of proteins. Many hundreds of different peptides have been isolated

from such hydrolyzates or synthesized by chemical procedures. Peptides are

also formed in the gastrointestinal tract during the digestion of proteins by

proteases, enzymes that hydrolyze peptide bonds. Peptides are named from

their component amino acid residues in the sequence beginning with the

amino-terminal ( abbreviated N-terminal) residue.

Much evidence supports the conclusion that the peptide bond is the sole

covalent linkage between amino acid in the linear backbone structure of

proteins. This evidence comes not only from chemical and enzymatic

degradation studies, but also from various physical measurements. For

example, proteins have absorption bands in the far ultraviolet (180 to 220

nm) and infrared regions that are similar to those given by authentic

peptides. Furthermore, x-ray diffraction analysis directly shows the presence

of peptide bonds in native proteins. There is only one other major covalent

linkage between amino acids: the disulfide bond of cystine serves in some

proteins as a cross-linkage between two separate polypeptide chains (

interchain disulfide bond) or between loops of a single chain( intrachain

disulfide bond ).

Peptides may be regarded as substituted amides. Like the amide group, the

peptide bond shows a high degree of resonance stabilization. The C―N

single bond in the peptide linkage has about 40 percent double-bond

204

character and the C=O double-bond about 40 percent single-bond character.

This fact has two important consequences: (1) The imino (―NH―) group of

the peptide linkage has no significant tendency to ionize or protonate in the

pH range 0 to 14.(2) The C―N bond of the peptide linkage is relatively rigid

and cannot rotate freely, a property of supreme importance with respect to

the three-dimensional conformation of polypeptide chains.

Chemical Properties of Peptides

The free N-terminal amino groups of peptides undergo the same kinds of

chemical reactions as those given by the α-amino groups of free amino

acids, such as acylation and carbamoylation. The N-terminal amino acid

residue of peptides also reacts quantitatively with ninhydrin to form colored

derivatives; the ninhydrin reaction is widely used for detection and

quantitative estimation of peptides in electrophoretic and chromatographic

procedures. Similarly, the C-terminal carboxyl group of a peptide may be

esterified or reduced. Moreover, the various R groups of the different amino

acid residues found in peptides usually yield the same characteristic

reactions as free amino acids.

One widely employed color reaction of peptides and proteins that is not given

by free amino acids is the burette reaction. Treatment of a peptide or protein

with Cu 2+ and alkali yields a purple Cu 2+ - peptide complex, which can be

measured quantitatively in a spectrophotometer.

1.3:1 Steps in the Determination of Amino Acid Sequence.

With this information on the properties of simple peptides as background, we

can examine the general strategy used to determine the amino acid

205

sequence of peptides and proteins devised by Frederick Sanger in 1953 in

his epoch-making determination of the amino acid sequence of the

polypeptide chains of insulin, the first protein for which the complete covalent

structure became known. Although each protein offers special problems, the

following sequence of steps are generally used:

1. If the protein contains more than one polypeptide chain, the individual

chains are first separated and purified.

2. All the disulfide groups are reduced and the resulting sulfhydryl groups

are alkylated.

3. A sample of each polypeptide chain is subjected to total hydrolysis, and

its amino acids composition is determined.

4. On another sample of the polypeptide chain the N-terminal and C-

terminal residues is identified.

5. The intact polypeptide chain is cleaved into a series of smaller peptides

by enzymatic or chemical hydrolysis.

6. The peptide fragments resulting from step 5 is separated, and their

amino acid composition and sequence are determined.

7. Another sample of the original polypeptide chain is partially hydrolyzed

by a second procedure to fragment the chain at points other than those

cleaved by the first partial hydrolysis. The peptide fragments are

separated and their amino acid composition and sequence are

determined.

8. By comparing the amino acid sequences of the two sets of peptide

fragments, particularly where the fragments from the first partial

hydrolysis overlap the cleavage points in the second, the peptide

fragments can be placed in the proper order to yield the complete

amino acid sequence.

206

9. The positions of the disulfide bonds and the amide groups in the

original polypeptide chain are determined.

Cleavage of disulfide bonds and separation of polypeptide chains

Before analyzing the amino acid sequence of a protein, the investigator must

determine whether the protein contains more than one polypeptide chain.

The number of chains is usually deduced from the number of N-terminal

amino acid residues per molecule of protein, by methods to be described

below. Clearly, the number of polypeptide chains will be equal to the number

of N-terminal amino acid residues per molecule of protein. If the polypeptide

chains have no covalent cross-linkages, they can be separated by treating

the protein with acid, base, or high concentrations of salt or a denaturing

agent.

If the polypeptide chains are covalently cross-linked by one or more disulfide

bonds between half residues of cystine, these cross-linkages must be

cleaved by appropriate chemical reactions. The commonest procedure is to

reduced the disulfide bond to sulfhydryl groups with an excess of

mercaptoethanol. An alkylating agent like iodoacetate is then used to alkylate

the sulfhydryl group of the cysteine residues to yield their S-carboxymethyl

derivatives. When the polypeptide chain is subsequently hydrolyzed, these

residues appear as S-carboxymethylcysteine, which is easily identified by the

chromatographic procedures used for amino acid analysis. Alkylation of

cysteine residues is desirable because of sulfhydryl group of cysteine is

relatively unstable and tends to undergo oxidation. Other reagents such as

iodoacetamide and ethyleneimine are also employed for alkylation of

sulfhydryl groups. An older and less common method for cleaving disulfide

cross-linkages, first developed by Sanger, is to oxidize the disulfide group to

yield cysteic acid residues from the half-cysteines. Once the interchain

207

disulfide bonds have been cleaved, the individual polypeptide chains are

separated, usually by eletrophoresis.

Even if the protein to be examined contains a single polypeptide chain, its

intrachain disulfide bonds, if any, must be cleaved and all cysteine residues

alkylated to the more stable S-carboxymethyl derivatives.

Fig - 1

Complete hydrolysis of polypeptide chains and determination of amino

acid composition

Once the polypeptide chain to be examined has been obtained in

homogeneous form, with no remaining disulfide cross-links or free sulfhydryl

groups, it is completely hydrolyzed and its amino acid composition

determined. Peptide bonds are readily hydrolyzed by heating with either acid

208

or base. Heating polypeptides with excess 6N hydrochloric acid at 100 to

120˚C for 10 to 24 h, usually in an evacuated, sealed tube, is the usual

procedure for complete hydrolysis. Little or no recemization of the amino

acids takes place under these conditions. However, not all the amino acids

are recovered quantitatively following acid hydrolysis; tryptophan is usually

destroyed by this treatment. Moreover, the amide groups of asparagines and

glutamine undergo complete hydrolysis in acid, to yield free aspartic and

glutamic acids, respectively, plus free ammonium ions.

Fig - 2

Polypeptides can also be hydrolyzed by boiling with strong sodium hydroxide

solutions, but alkaline hydrolysis causes destruction of cysteine, serine, and

threonine and recemization of all the amino acids. Alkaline hydrolysis is

normally used only for the separate estimation of tryptophan, which is

unstable to acid but stable to base.

209

The amino acid composition of hydrolyzates of polypeptides and proteins is

determined by automated ion-exchange chromatography in an amino acid

analyzer. The first pure protein for which the complete amino acid

composition was deduced was β-lactoglobulin of milk. This analysis, which

required several years of work by older methods, was completed in 1947.

Today, the amino acid analyzer determines the complete amino acid

composition of a protein hydrolyzates within 2 to 4 hrs. for which very small

samples are required.

At this point it is instructive to consider the amino acid composition of

representative pure proteins. Some generalizations may be made from these

and other available data:

1. Not all proteins contain all the 20 amino acids normally found in

proteins; e.g., ribonuclease lacks tryptophan. Fibrous proteins, e.g., silk

fibroin and collagen, lack several amino acids.

2. Some amino acids occur much less frequently in proteins than others.

For example, in most proteins there are relatively few histidine,

tryptophan, and methionine residues.

3. In most proteins 30 to 40 percent of the residues are amino acids with

nonpolar R groups. Membrane proteins tend to have a somewhat

higher content. Over 90 percent of the amino acid residues of the

insoluble fibrous protein elastin, are nonpolar.

4. In some proteins, such as lysozyme, cytochrome c, and the histones,

the positively charged R groups predominant (at pH 7.0); such proteins

are basic. In others, the negatively charged R groups of glutamic or

aspartic acid predominate, as in pepsin, which is highly acidic.

Identification of the N-terminal residue of a peptide

210

Very important in the procedure for establishing amino acid sequence are

methods for identifying the terminal amino acid residues. The first useful

method for the N-terminal residue of polypeptides was described by

Sanger, who found that the free unprotonated α-amino group of peptides

reacts with 2,4-dinitrofluorobenzene (DNFB) to form yellow 2,4-

dinitrophenyl derivatives. When such a derivative of a peptide, regardless

of its length, is subjected to hydrolysis with 6 N dinitrophenyl derivative

HCl, all the peptide bonds are hydrolyzed, but the bond between the 2,4-

dinitrophenyl group and the α-amino group of the N-terminal amino acid is

relatively stable to acid hydrolysis. Consequently, the hydrolyzates of such

a dinitrophenyl peptide contains all the amino acid residues of the peptide

chain as free amino acids except the N-terminal one, which appears as

the yellow 2,4-dinitrophenyl derivative. This labeled residue can easily be

separated from the unsubstituted amino acids and identified by

chromatographic comparison with known dinitrophenyl derivatives of the

different amino acids.

211

Fig.- 3

Sanger's method has been largely supplanted by more sensitive and

efficient procedures. One employs the labeling reagent 1-

dimethylaminonaphthalene-5-sulfonyl chloride ( abbreviated dansyl

chloride ), as shown in figure 5-7. Since the dansyl group is highly

fluorescent, dansyl derivatives of the N-terminal amino acid

212

Can be detected and measured in munite amounts by fluorimetric

methods. The dansyl procedure is 100 times more sensitive than the

Sanger method.

The most important and most widely used labeling reaction for the N-

terminal residue is that designed by P.Edman. In the Edman procedure

phenylisothiocyanate reacts quantitatively with the free amino group of a

peptide to yield the corresponding phenylthiohydantoin derivatives, which

can be separated and identified, usually by gas-liquid chromatography.

Alternatively, the N-terminal residue removed as the phenylthiocarbamoyl

derivative can be identified simply br determining the amino acid

composition of the peptide before and after removal of the N-terminal

residue; this is called subtractive Edman method.

The great advantage of the Edman method is that the rest of the peptide

chain after removal of the N-terminal amino acid is left infact for further

cycles of this procedures; thus the Edman method can be used in a

sequential fashion to identify several or even many consecutive amino

acid residues starting from the N-terminal end. This great advantage has

been further exploited by Edman and G. Begg, who have perfected an

automated amino acid "sequenator" for carrying out sequential

degradation of peptides by the phenylisothiocyanate procedure.

Automated amino acid sequencers, now widely used, permit very rapid

determination of the amino acid sequence of peptides up to 20 residues.

213

Fig - 4

In some native proteins the N-terminal residue is buried deep within the

tightly folded molecule and is inaccessible to the labeling reagent; in such

cases denaturation of the protein can render it accessible. In other

proteins, e.g., the tobacco mosaic virus coat protein, the α-amino group of

the N-terminal amino acid is acetylated and hence not reactive to labeling

reagents. Some natural peptides have no free N-terminal α-amino group

because they are cyclic; e.g., the antibiotic tyrocidin A has 10 amino acid

residues in a circular arrangement (figure 5-2). However, there is no

evidence that circular polypeptide chains occur in proteins.

214

Identification of the C-terminal residues of peptides

The C-terminal amino acid of peptides can be reduced with lithium

borohydride to the corresponding α-amino alcohol. If the peptide chain is

then completely hydrolyzed, the hydrolyzates will contain one molecule of

an α-amino alcohol corresponding to the original C-terminal amino acid.

This can be easily identified by chromatographic methods; all the other

residues will be found as free amino acids.

215

Fig – 5

Another important procedure is hydrazinolysis (figure 5-9), which cleaves all

the peptide bonds by converting all except the C-terminal amino acid

residues into hydrazides. The C-terminal residue appears as a free amino

acid, which can be readily identified chromatographically.

216

Fig. - 6

C-terminal amino acid of a peptide can be also be selectively removed by

action of the enzyme carboxypeptidase, which specifically attacks C-

terminal peptide bonds. A drawback is that the enzyme, after removal of

the terminal residue, proceeds to attack the new C-terminal peptide bond.

It is therefore necessary to measure the rate of liberation of different

amino acids from the peptide by carboxypeptidase in order to identify the

C-terminal residue unequivocally.

1.4 STRUCTURE OF PROTEINS

1.4:1 Forces responsible for structure of proteins

217

The following types of bonds play an important role in the formation of

proteins.

1.Peptide bond and peptides

The peptide bonds helps in the formation of primary structure of proteins. A

single

peptide bond is formed when two amino acids are involved in a reaction and

the

carboxyl group(-COOH) of one amino acid reacts with the amino group (-

NH2) of

another amino acid, with the elimination of one molecule of water. The bond

between the two amino acids (-CO-NH-)is called peptide bond and the

compound formed by the condensation of amino acids is known as a

dipeptides.

H H H H | | | |

R―C― CO OH + H NH―C―R‟ → R― C― CO―NH ― C ―R‟ | | ―H2O | peptide bond | NH2 COOH NH2 COOH Dipeptides

Formation of peptide linkage

Depending upon the number of amino acids involved in a reaction, the

compound is known as a dipeptides (two amino acids with one peptide

bond), a tripeptides (three amino acids with two peptide bonds), a

tetrapeptide (four amino acids with three peptide bonds) or a polypeptide

[many (n….) amino acids with n-1 peptide bonds] where n=number of amino

acids. When a polypeptide chain is formed, one free amino and one free

amino and one free carboxyl group is left at the two different ends. The end

having free amino group is referred as amino terminal or N-terminal, while

the end having free carboxyl group is called carboxy terminal or C-terminal.

218

2. Disulphide bond

Disulphide bond is also characteristic of the primary structure of proteins. It is

a covalent bond and is generally established between cysteine residues as

in insulin and in ribonucleases.The disulphide bond may also be established

in other sulphur containing amino acids like Cystine and methionine. When

the thiol groups of two cysteine molecules are reversibly oxidized, they

form the disulphide compound, cysteine, and the linkage established

between them is known as disulphide (―S―S―) linkage. The

disulphide bond may be formed either within the single polypeptide chain

(intramolecular) or between the two polypeptide chains

(intermolecular).Disulphide bond helps in stiffening the folded polypeptide

chain and also in joining two or more polypeptide chains together.

HS― CH2―CH―COOH S ―CH2―CH―COOH | |

NH2 NH2

HS― CH2―CH―COOH S ―CH2―CH―COOH | |

NH2 disulphide bond NH2

2 molecules of cysteine cysteine

Formation of disulphide bond

3.Hydrogen bonds

Hydrogen bonds are commonly found among the proteins. They are

electrostatic in origin and reflect the interaction of the incompletely shielded

nucleus of the hydrogen atom―which is a portion of unit positive charge,

with the electronic system of another atom. The hydrogen bonds are formed

by only electronegative atoms. This bond results by sharing of electrons

between hydrogen atom and other electronegative atoms like oxygen.

1.Hydrophobic bond

219

Hydrophobic bonds arise from the mutual cohesion of non-polar hydrocarbon

side chains. In biological systems there are a number of amino acids having

side-chains which are of hydrocarbon nature. These are hydrophobic groups

in that they do not form hydrogen bonds with water molecules. On the other

hand, water molecules have a strong tendency to form hydrogen bonds

among themselves. As a result of the hydrogen bonding among water

molecules, hydrocarbons are forced out of any water phase in which they

may be placed. Similarly, hydrocarbon side-chains of the various amino acids

tend to be forced together as a result of the hydrogen bonding

among water molecules. The hydrophobic bonds are believed to contribute

most of the structural stabilization energy for the majority of proteins.

Primary, Secondary, Tertiary & Quarternary Structure of Proteins

The sequential arrangement of the amino acids in a protein molecule is

known as the primary structure. When an interaction between

polypeptide takes place, it gives rise to a helical type structure known as

secondary structure. Further folding and coiling gives rise to the highly

specific and complex tertiary and quarternary structures

Primary Structure

The sequential arrangement of the various amino acids in a protein (

Polypeptide chain ) through the peptide bonds is known as the primary

structure. Each protein molecule consists of one or more polypeptide chains

in which the amino acids are linked by peptide linkages. Myoglobin, a protein,

consists of only single polypeptide chain, whereas hemoglobin molecule

consists of four polypeptide chains. The covalent bonds and disulphide

(―S―S―) bonds are again characteristics of the primary structure of

proteins. The disulphide bond is generally established between cysteine

residues, as in insulin and ribonucleases.If the protein has only one

220

polypeptide chain, it can have only one free α-amino group(-NH2 terminal)

and one free carboxyl (-C-terminal) group. In the determination of primary

structure of protein, it is essential to know what amino acids are a N-terminal

and C-terminal ends. The free α-amino group can react with the

reagents like dinitrofluorobenzenes to give a dinitrophenyl (DNP)

derivative which on hydrolysis yields the yellow coloured DNP –amino acids.

These can be isolated chromatographically.

Secondary Structure of Protein

The covalent backbone of a polypeptide chain is formally single-bonded. We

would therefore expect the backbone of a polypeptide chain to have an

infinite number of possible conformations and the conformation of any given

polypeptide to undergo constant change because of thermal motion.

However, it is now known that the polypeptide chain of a protein has only one

conformation (or a very few) under normal biological conditions of

temperature and pH. This native conformation, which confers biological

activity, is sufficiently stable so that the protein can be isolated and retained

in its native state. This fact therefore impiles that the single bonds in the

backbone of native proteins cannot rotate freely. When the long polypeptide

chains in a protein undergo folding, they form the secondary structure or

helical structure. The secondary structure is determined by hydrogen

bonding between the components of the peptide chain itself. The hydrogen

bonds can occur either within one polypeptide chain or between different

polypeptide chains of the protein molecule.Thus, the secondary structure of

proteins is represented by helical structures which are ultimately formed by

the hydrogen bonding between the chains or chain.

Fine Structure of Proteins

221

α-Structure: Fine structure of proteins was discovered by Pauling and Corey

(1951) of California Institute of Technology and were awarded Nobel Prize in

chemistry for this discovery. They proposed the α-helical conformation of

protein based on theoretical grounds.

β-Structure : Asbury and Street (1933) proposed β-Structure of proteins

which was later modified by Pauling and Corey. The β-structure is

represented by parallel zig-zag polypeptide chain which form a pleated

sheet-like structure. The hydrogen bonds are formed between NH and C=O

groups on the neighboring chains which stabilize the β-structure of proteins.

The side chain attached to the amino acid residues lies above and below the

hydrogen bonded sheets. This structure is a stable arrangement where side

chains are small and do not cause distortion of the pleated structure. In

fibroin, the chains run anti-parallel to each other, i.e the free amino (or

carboxyl) groups are at opposite ends of neighbouring chains. β-Structure is

found in milk and keratin.

Fibrous Proteins

We shall consider the conformation of fibrous proteins first. Not only are they

very abundant, particularly in higher animals, but they also have simpler

conformations than the globular proteins, since their polypeptide chains are

usually arranged or coiled along a single dimension, often in parallel bundles.

As a result the conformation of the polypeptide chains in some fibrous

proteins has been easier to examine experimentally; actually, the fibrous

proteins gave the first important clues to the constraints on the freedom of

rotation of the single bonds in the polypeptide-chain backbone of

proteins.Two major classes of fibrous proteins, the keratins and collagens,

will be considered here. Study of the keratins has been especially important

222

in revealing the most prevalent conformations of the polypeptide chains in

native proteins, namely, the α-helix and β conformation.

The Keratins

The Keratins are fibrous, insoluble proteins of animals derived from

ectodermal (skin) cells. They include the structural protein elements of skin (

leather is almost pure keratin) as well as the biological derivatives of

ectoderm, such as hair, wool, scales, feathers, quills, nails, hoofs, horns, and

silk. There are two classes of keratins. The α-keratins are relatively rich in

cystine residues and thus contain many disulfide cross bridges; in addition,

they contain most of the common amino acids. The α-keratins include the

hard, brittle proteins of horns and nails, which have a very high content of

cysteine (up to 22 percent), as well as the softer, more flexible keratins of

skin, hair, and wool, which contain about 10 to 14 percent cystine. The β–

keratins, on the other hand, contain no cysteine or cysteine but are rich in

amino acids with small side chains, particularly glycine, alanine, and serine.

The β–keratins are found in the fibers spun by spiders and silkworms and in

the scales, claws, and beaks of reptiles and birds. Another important

difference is, that the α-keratins stretch when heated; hair, for example,

stretches to almost double its length when exposed to moist heat but

contracts to its normal length on cooling. The β–keratins do not stretch under

these conditions. Electron microscopy has revealed that hair and wool fibers

contain bundles of macrofibrils, each made up of thinner fibrils consisting in

turn of parallel bundles of protein filaments arranged along a single axis. This

structural feature allows them to be examined readily by x-ray diffraction

analysis.

The α-Helix and the Structure of α-Keratins

223

Pauling and Corey used precisely constructed models to study all the

possible ways of twisting or coiling the backbone of the polypeptide chain

along one axis, in view of the constraint imposed by the planar peptide

bonds, to account for the observed repeat units of 0.50 to 0.55 nm in α-

keratins. The simplest arrangement they found is the helical structure shown

in the figure 6-5. In this structure, the α-helix, the backbone is arranged in a

helical coil having about 3.6 amino acid residues per turn. The R groups of

the amino acids extend outward from the rather tight helix formed by the

backbone. In such a structure the repeat unit, consisting of a single complete

turn of the helix, extends about 0.54 nm,(5.4 Å) along the long axis,

corresponding closely to the major periodicity of 0.50 to 0.55nm deduced

from the x-ray pattern of natural α-keratins. The rise per residue is about 0.15

nm, corresponding to the minor periodicity of 0.15 nm also observed in the

diffraction patterns. Such an α-helix permits the formation of intrachain

hydrogen bonds between successive coils of the helix, parallel to the long

axis of the helix and extending between the hydrogen atom attached to the

electronegative nitrogen of one peptide bond and the carbonyl oxygen of the

third amino acid beyond it. The electrical vectors of these hydrogen bonds

are so oriented that they give nearly maximal bond strength. But especially

significant is that the α-helical arrangement allows every peptide bond of the

chain to participate in intrachain hydrogen bonding. Although other kinds of

helical coils of polypeptide chains can be formed, such as a π helix (4.4

residues per turn), they cannot account for the characteristic spacing of the

repeat units in the α-keratins family of proteins, nor would they be as stable

as the α-helix.

An α-helix may form with either L-or D-amino acids, but a helix cannot form

from a polypeptide chain containing a mixture of L and D residues.

224

Furthermore, starting from the naturally occurring L-amino acids, either right-

handed or left-handed helical coils can be built; however, the right-handed

helix is significantly more stable. In all native proteins examined to date, the

α-helix is right-handed. From these structural considerations Pauling and

Corey proposed that the α-keratins consist of polypeptide chains in right-

handed α helical coils. In the α-keratins of hair and wool, three or seven such

α helixes may be coiled around each other to form three-stranded or seven-

stranded ropes (figure 6-6), held together by disulfide cross-linkages. The α-

helix represents the secondary structure of α-keratins, i.e., the regular, coiled

conformation of their polypeptide chains around and along their long axis.

β -keratins: The β Conformation and the Pleated Sheet

We have seen that x-ray study of α-keratins led to our present

knowledge of the α-helix; in a similar way, x-ray studies of β–keratins have

revealed important clues to the β conformation of the polypeptide chain. We

recall that when fibers of α-keratins are subjected to moist heat, they can be

stretched to almost double their original length. In this stretched condition

they yield x-ray diffraction patterns resembling that of silk fibroin, an example

of a β–keratin. Pauling and Corey concluded that the transition from α-keratin

to β–keratin structure when hair or wool is steamed is caused by the thermal

breakage of the intrachain hydrogen bonds that normally stabilize the α-helix

and the consequent stretching of the relatively tight α-helix into a more

extended, zigzag conformation of the polypeptide chain, characteristic of β–

keratins generally, which they designated the β conformation. Side-by-side

polypeptide chains in the β conformation are arranged in pleated sheets,

which are cross-linked by interchain hydrogen bonds. All the peptide linkages

participate in this cross-linking and thus lend the structure great stability; the

R groups lie above or below the zigzagging planes of the pleated sheet. This

225

is the type of secondary structure found in fibroin secreated by the silkworm

Bombyx mori. In most types of fibroin every other amino acid is glycine, so

that all the R groups on one side of the pleated sheet are hydrogen atoms.

Since alanine makes up most of the rest of the amino acids of fibroin, most of

the R groups on other side of the sheet are methyl groups. Fibroin and other

β–keratins are rich in amino acids having relatively small R groups,

particularly glycine and alanine. If the R groups are bulky or have like

charges, the pleated sheet cannot exist because of R group interactions.

This is why the stretched form of α-keratins is unstable and reverts

spontaneously to the α-helical form; the R groups of α-keratins are bulkier

and more highly charged than those of silk fibroin.

There are two other differences between α-keratins and native β–keratins. In

the α forms all the polypeptide chains are parallel, i.e., run in the same N-

terminal to C-terminal direction, whereas in fibroin, the adjacent polypeptide

chains are antiparallel, i.e., run in opposite directions. Also, α-keratin

contains many cystine residues so arranged as to provide interchain

―S―S― cross-linkages between adjacent polypeptide chains. In contrast,

the β–keratins, such as fibroin, have no ―S―S― cross-linkages.

Collagen

Another major type of fibrous protein in higher animals, the collagen of

connective tissues, is the most abundant of all proteins in higher vertebrates,

making up one-third or more of the total body protein. The larger and heavier

the animal, the greater the fraction of its total proteins contributed by

collagen. It has been aptly said, for example, that a cow is largely held

together by the collagen fibrils in its hide, tendons, bones, and other

connective tissues. Collagen fibrils are arranged in different ways, depending

on the biological function of the , particular type of connective tissue. In

226

tendons collagen fibers are arranged in parallel bundles to yield structures of

great strength but little or no capacity to stretch. In the hide of the cow the

collagen fibrils form an interlacing network laid down in sheets. The organic

material of the cornea of the eye is almost pure collagen. Whatever the

arrangement of collagen fibrils in connective tissue, the fibrils always show a

characteristic cross-striated appearance under the electron microscope, in

which the repeat distance is between 60 and 70 nm, depending on the type

of collagen and spices of organism. Boiling in water converts collagen into

gelatin, a mixture of polypeptides.

Collagens also have a distinctive x-ray diffraction pattern, different from

those of α- and β-keratins. From comparisons of the x-ray patterns of

collagen and of polyproline it has been deduced that the secondary structure

of collagens is that of a triple helix of polypeptide chains. Each of the chains

is a left-handed three-residue helix; the chains are held together by hydrogen

bonds. The frequent praline residues determine the distinctive type of helical

arrangement of the chain, whereas the smaller R groups of the glycine

residues, which occur in every third position, allow the chains to intertwine.

The complete amino acid sequence of the collagen chains is not yet known,

but –Gly-X-Pro-. –Gly- Pro-X-, and –Gly-X-Hyp- are frequently occurring

sequences, in which X may be any amino acid. No proteins other than the

collagens appear to contain similar triple-helical chains.

Collagens is built of recurring subunit structure, triple–standard tropocollagen

molecules, having distinctive"heads". These subunits are arranged heads to

tail in many parallel bundles, but the heads are staggered, thus accounting

for the characteristic 60-to 70-nm spacing of the repeat units in collagen

fibrils from different species. The polypeptide chains of tropocollagen are

covalently cross-linked by dehydrosinonorleucine residues, formed by an

227

enzymatic reaction between two lysine residues of adjacent tropocollagen

subunits.

The secondary structure of the polypeptidechains in other fibrous proteins is

not yet known. Studies are under way on elastin of the elastic connective

tissue of ligaments and on sclerotin, the structural protein of the light, rigid

exoskeleton of insects. Elastin is especially interesting since its polypeptide

chains are covalently connected to form a stretchy, two-dimensional sheet

resembling a trampoline net. The polypeptide chains are joined through

covalent attachment to residues of demosine and isodemosine . Another

structural protein of great interest, resilin, found in the wing hinges of some

insects, is remarkable for its perfectly reversible elastic properties.

Tertiary Structure of Globular Proteins

Very few protein molecules exist as a simple α-helix. Further degrees of

folding or coiling of polypeptide chains in α-helix give a complex three-

dimensional structure (tertiary structure) which often contains helical and

non-helical regions Folding of the α-helix occurs where the amino acid

proline has an imino group instead of an amino group which causes

unstability in the α-helix by producing hindrance in the regular internal

hydrogen bonding. Three main types of bonds, ionic, hydrogen and

hydrophobic, are responsible for the formation of the tertiary structure of a

protein. Dipole-dipole interaction and disulphide linkages are also

responsible for the formation of tertiary structure. Tertiary structure of

proteins is probably thermodynamically the most stable and is of most stable

and is of much importance, because the enzymatic properties of a protein

depend on it.

228

Types of interaction which may contribute to the stabilization of protein structure. (a) Electrostatic bonds (b) hydrogen bonds, (C and D) Hydrophobic bonds, (e)

disulphide linkage.

Fig. - 7 We now turn from the fibrous proteins, which have relatively simple

structures, to the far more complex globular proteins, which have polypeptide

chains tightly folded into compact three-dimensional structures with many

different kinds of specialized biological activities.

Until x-ray analysis of crystalline globular proteins became feasible, next to

nothing could be learned about how their polypeptide chains are folded in

three dimensions. In fact , only the barest outlines of the shape of the

globular proteins can be deduced from other physical methods, e.g.,

measurement of viscosity, sedimentation, and diffusion, which allow

calculation of the axial ratio of protein molecules but can give no information

on their internal structure.

Interpretation of the x-ray diffraction patterns is far more difficult for globular

than for fibrous proteins because the polypeptide chains of globular proteins

are not arranged along one axis but are irregularly and compactly folded into

nearly spherical shapes. However, the introduction of intensely diffracting,

229

electron-dense heavy-metal atoms into the molecules of globular proteins to

provide reference points for the mathematical interpretation of the diffraction

patterns has made it possible to determine the three-dimensional structures

of a number of globular proteins to a resolution of 0.6 nm, and in cases 0.2

nm. Among the globular proteins whose tertiary structures are now well

known are trypsin, carboxypeptidase A, cytochrome c, lactate

dehydrogenase, and subtilisin, a proteolytic enzyme from a bacterium.

Although the conformations of only a few proteins are known in detail, the

results have already yielded some important generalizations that are

probably applicable to many globular proteins.

The Stabilization of Tertiary Structure of Globular Proteins

Once the native tertiary structure of a globular protein has formed, four

mojor types of week interactions or bonds cooperate in stabilizing it: (1)

hydrogen bonds between peptide groups, as in α-helical or β–pleated sheets;

(2) hydrogen bonds between R groups; (3) hydrophobic interactions between

nonpolar R groups; and (4)ionic bonds between positive charged and

negatively charged groups, such as the―COO- of aspartate or glutamate R

groups and the ―NH3+ of lysine R groups. From studies on the relative

contribution of each of these four types of weak bond to the total

conformational stability of native protein molecules it is now clear that

hydrophobic interactions between the nonpolar R groups are by far the most

important. Most proteins contain from 30 to 50 percent of amino acids with

nonpolar R groups; x-ray analysis shows that nearly all these R groups in the

interior of native globular proteins, shielded from exposure to water.

To fully understand the important role of hydrophobic interactions in

stabilizing protein structure, we must ask a fundamental question: Why does

a denatured, randomly coiled polypeptide chain tend to fold spontaneously

230

into a highly ordered, biologically active conformation, a process that

apparently decreases the entropy of the polypeptide chain? Is protein folding

a violation of the second law of thermodynamics, which states that all

processes proceed in that direction which maximizes entropy, or

randomness? The answer of this dilemma is found in a balance of forces.

One force is the tendency of the polypeptide chain to seek its own

conformation of maximum randomness or entropy. The opposing force is the

tendency of the surrounding water molecules to seek their position of

maximum randomness or entropy. The critical factor in this balance of forces

is represented by the nonpolar R groups. When nonpolar groups are

inserted into water, a new interface is created, which requires the adjacent

water molecules to assume a more ordered arrangement than they would

have in pure liquid water; thus input of energy is required to force a nonpolar

R group into water. A random polypeptide chain,with its nonpolar R groups

exposed, will thus tend to assume a conformation in which the nonpolar R

groups are shielded from exposure to water. It is the tendency of the

surrounding water molecules to relax into their maximum-entropy state that

brings about the transition of the plypeptide chain from a random unfolded

state to a highly ordered tertiary conformation. At equilibrium, when the

random chain is fully folded, the increase in the entropy of the surrounding

water molecules is greater than the decrease in the entropy of the now

correctly coiled polypeptide chain. The second law has not been violated

because the combination of the system (the polypeptide) and the

surroundings (the water) has undergone a net increase in entropy. However,

much evidence suggests that the folded, native conformation is more stable

than the unfolded, or denatured, conformation by only a relatively small

margin. The stability of a native globular protein is thus the result of a

231

delicate balance between two relatively massive and opposing forces:(1) the

tendency of the polypeptide chain to unfold into a more random arrangement

and(2) the tendency of the surrounding water molecules to seek their most

random state.We have assumed in this discussion that the native

conformation of a globular protein is more stable. i.e., has less free energy,

than the random-coil from under biological conditions, but this assumption

may not be true for all proteins; it is the subject of much debate and study.

Proteins that spontaneously refold into their native from may indeed be more

stable than their denatured forms under specific conditions of pH, ionic

strength, and temperature. On the other hand, the unfolded form of some

proteins may have less free energy than the native form. In such cases the

transition from the native to the random state may have a very high

activation-energy barrier, thus locking the polypeptide chain into its native

conformation. In this case, once the native form is unfolded, the polypeptide

chain will not spontaneously refold into the native conformation.

The Quaternary Structure of Oligomeric Proteins

This defines the degree of polymerization of a protein unit. The quarternary

structure is exhibited by haemoglobin molecule which was determined by

Perutz and coworkers (1960). They showed that this protein undergoes

further organization, being made up of 4-polypeptide chains. This further

organization is known as the quarternary structure. The chains undergo

secondary folding, two of the structures consisting of α-chain and other two

of β-chain. All 4-chains fit together in a compact tetrahedral arrangement to

form a complete haemoglobin molecule. The forces maintaining the

quarternary structure are similar to those involved in tertiary structure, but the

association of the sub-units, in general is more flexible.

232

We have seen that some globular proteins are oligomeric, i.e., contain two or

more separate polypeptide chains or subunits. The quaternary structure

designates the characteristic manner in which the indivisual, folded

polypeptide chains fit each other in the native conformation of an oligomeric

protein. Among the simplest oligomeric proteins is hemoglobin, which has

four polypeptide chains.

Because oligomeric protein have relatively high molecular weights, and

because they contain multiple chains each of which may have a

characteristic conformation, their three-dimensional conformation is far more

difficult to analyze by x-ray methods than that of single-chain proteins.

Haemoglobin

Haemoglobin was the first oligomeric protein for which the complete tertiary

and quaternary structure became known from x-ray analysis. This

achievement, accomplished by M.F.Perutz and his colleagues in England,

culminated some 25 years of detailed study of the structure of this important

protein. Because of the similarity of function and the homology of amino acid

sequence of the polypeptide chains of myoglobin and hemoglobin, a number

of extremely important relationships have developed from concurrent

investigations of the structure of these two proteins, which were carried out in

the same laboratory.

Haemoglobin contains two α chains and two β chains, to each of which is

bound a heme residue in noncovalent linkage. The molecule was examined

in its oxygenated form, which has a compact spheroidal structure of

dimensions 6.4 by 5.0 nm. Figure 6-19 shows the low-resolution outlines of

the haemoglobin chains, and figure 6-20 shows how the chains fit together in

an approximately tetrahedral arrangement. Each chain has an irregularly

folded conformation, in which lengths of pure α-helical regions are separated

233

by bends. Both the α and β chains have about 70 percent α-helical

chapter, as is true for myoglobin. The α and β chains are very similar to each

other in their tertiary structure, which consists of similar lengths of α helix

with bends of about the same angles and directions. But most remarkable is

that the tertiary structure of the α and β chains is very similar to that of the

single chain of myoglobin, consonant with the similar biological function of

these two proteins, namely, their capacity to bind oxygen reversibly,

myoglobin in muscle and haemoglobin in blood.

In haemoglobin there is very little contact between the two α chains and

between the two β chains, but there are numerous R-group contacts

between the pairs of unlike chains. Of special interest is the location of the

four heme groups, one in each subunit, that bind the four molecules of

oxygen. These heme groups, flat molecules in which the iron atoms forms

square-planar co-ordination complexes, are quite far apart from each other

and are situated at different angles from each other. Each is partially buried

in a pocket lined with nonpolar R groups. The fifth coordination bond of each

iron atom is to an imidazole nitrogen of a histidine residue; the sixth position

is available for co-ordination with an oxygen molecule, lined with polar R

groups.

The amino acid sequences of hemoglobin chains of many species have been

compared. Although only nine of the residues in each chain are absolutely

invariant, the amino acid replacements in many other positions suggest that

the polypeptide chain subunits of the haemoglobins from nearly all species

have the same tertiary structure. Moreover, in nearly all haemoglobins a

histidine R group co-ordinates with the iron atom of the heme group.

234

The quaternary conformation of other oligomeric proteins has now been

established, in particular the enzyme lactate dehydrogenase, which also has

four polypeptide chains.

Check your progress- 1

Note:-

1. Write your answer in the space given below.

2. Check your answer with the one at the end of the unit.

Q 1. Write short notes on

a) α helix b) β-conformation c) Structure of

haemoglobin

Q.2. Write a note on amino acid sequencing.

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

235

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

236

--------------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------------

1.5 AMINO ACID METABOLISM

1.5:1 Biosynthesis of amino acids. The lower animals are capable of

synthesizing all the amino acids present in proteins from the amphibolic

intermediates. On the other hand, the higher animals can‟t synthesize certain

amino acids in adequate quantities and therefore such amino acids must be

taken in the diet. These are the nutritionally essentials amino acids and thus

those which can be synthesized from amphibolic intermediates are known as

nutritionally non-essentials amino acids.

Biosynthesis of non-essential amino acids.

1. L-Glutamate α-ketoglutarate; the reaction is catalyzed by L-glutamate

dehydrogenase. Although the reaction is reversible, the equilibrium

constant favours glutamate formation.

α-ketoglutarate + NH2+ NAD(P)H + H+←→ L-Glutamate + NAD+(P)+

2. Glutamine. It is synthesized from glutamate and ammonia molecule in

presence of glutamine synthetase, a mitochondrial enzyme ( present in

highest amounts in renal tissue) which requires ATP and Mg++ ions.

NH2+ L-Glutamate+ ATP Glutamine synthetase L-Glutamine + ADP+ Pi

Mg++

3. Proline. It is also synthesized from glutamate by the reversal of the

catabolic route of praline to glutamate. Hydroxyproline is derived from

praline by an oxygenase reaction; the substract being a prolyl-

containing polypeptide and oxygen is supplied air.

4. Alanine and aspartate. These are formed by the transamination

reactions of pyruvate and oxaloacetate respectively. Like glutamine,

237

asparagine is obtained from aspartate and ammonia in a reaction

catalysed by asparagines synthetase.

5. Tyrosine. It is obtained by the hydroxylation of phenylalanine ( an

essential amino acid) in presence of phenylalanine hydroxylase.

6. Cysteine. As discussed earlier, it is synthesized from methionine

(essential) and serine(non-essential) ; the former supplies sulphur by

transulfuration, and the latter supplies the carbon skeleton.

7. Serine. It has been shown to be obtained from D-3 phosphoglycerate

(an intermediate in glycolysis) by two different routes. Of the two routes

one involves the phosphorylated intermediates and the other involves

non-phosphorylates. The three important reactions in both the routes

are same, viz. oxidation, transmination and hydrolysis although with

specific sequence which is clear from the following figure no.

238

Fig. - 8

It is probable that the route involving phosphorylated intermediates

accounts for most of the serine synthesized by mammalian tissues.

8. Glycine. In mammalian tissues, it can be synthesized by three different

routes.

239

a. From glyoxalic acid: The cytosol of liver tissue contains active glycine

transaminase which catalyses the transamination of glyoxalate with

glutamate to glycine.

COOH COOH

| | CHO CH.NH2 Glycine CH2 NH2 C O | + | transaminase | + | COOH (CH2)2 COOH (CH2)2 | | COOH COOH

Glyoxalate Glutamate Glycine α-ketoglutarate

b. From serine: Serine, ammonium ions, bicarbonate, tetrahydrofolate

(FH4), pyridoxal phosphate (PLP), and a source of reducing power

(NADH) condense in presence of an enzyme system found in the liver

mitochondria of mammals to form two moles of glycine.

CH2OH | FH4 CH2. NH2 CH.NH2 + CO2 + NH2 + 2H PLP 2 | + H2O | COOH COOH serine Glycine

c. From choline: Choline (derived from serine) may also be converted

into glycine in the following way (see also catabolism of serine).

+ choline oxidase + CH2OH.CH2.N.(CH3)2 COOH. CH2.N.(CH3)2 ( -2H)

Choline Betaine aldehyde NDA+ Betaine aldehyde (-NADH,-H+) dehydrogenase + COOH. CH2.N.(CH3)2 Demethylation COOH. CH2.N.(CH3)2 (-CH3) Dimethylglycine Betaine Dimethylglycine oxidase (- CH2O) Sarcosine oxidase COOH.CH2.NH.CH3 COOH.CH2.NH2

240

Sarcosine (- CH2O) Glycine

d. In clostridia, not in mammals, L-threonine or L-allothreonine undergoes

aldol type cleavage to form glycine and acetaldehyde.

COOH COOH | | COOH

H2N― C ―H Or H― C ― NH2 → | + CH3CHO | | CH2. NH2 H―C―OH O―C―H | | CH3 CH3 L-Threonine L-Allothreonine Glycine

9. 5-Hydroxylysine. It is formed by the hydroxylation of lysine. The

mechanism of hydroxylation has been extensively studied in

developing chick embryo. The probable mechanism is as below.

CH2. NH2 CH2. NH2

| | CH2 CHOH

| | CH2 O2[H] CH2 | ―H2O | CH2 CH2 | | CH. NH2 CH. NH2 | | COOH COOH Lysine 5-Hydroxylysine

5-Hydroxylysine is found to be present in collagen and collagen products

such as gelatin.

1.5:2 GENERAL CATABOLISM OF AMINO ACIDS.

Although several amino acids pursue individual catabolic pathways, a few

general reactions are found to be common in the catabolism of nearly all the

amino acids. With few exceptions, the catabolic pathway of amino acids

begins with their conversion to α-keto acids. The amino group, eliminated in

the form of ammonia, joins the ammonia pool either as such or in the

combined form. The majority of α-keto acids produced from amino acids join

241

the carbohydrate metabolism, while a minority is more closely related to the

ketone bodies and fatty acids. However, a few amino acids do not undergo

catabolism in this way but they behave in different individualistic way, they

will be discussed separately.

Conversion of α-amino acids to α-keto acids

There are two important ways by which amino acids are converted into their

corresponding α-keto acids and thus the amino group is removed from the

carbon skeleton in the form of ammonia.

1.Oxidative deamination: In oxidative deamination, the amino acids lose

hydrogen atoms to produce an imino acid which is then hydrolysed to

ammonia and a keto acid.

R R R | | +2H2O | CH.NH2 C=NH CO + NH3 | | | COOH COOH COOH Amino acid Imino acid Keto acid

The reaction is catalysed by the amino acid oxidase and the coenzyme (FAD

or FMN) which takes up hydrogen. Amino acid oxidase are of two types

depending upon the nature of the substrate on which they act. One of these

is the L-amino acid oxidase which attacks most of the L-amino acid. The L-

amino acid oxidase contains FMN as hydrogen acceptor and is found mainly

in liver and kidney. But even in these organs its concentration is too less to

be of any significant importance. The other type of amino acid oxidases are

specific for D-amino acids and hence known as D-amino acid oxidases.

These contain FAD as the hydrogen acceptor and are of wide occurrence in

animal tissues. Owing to their high concentration, they are quite active but

their action is limited owing to non-availability of D-amino acids in the

organisms.

242

Oxidative deamination of glutamic acid may also be catalysed by an

important enzyme, the L-glutamate dehydrogenase. This enzyme is highly

active and found abundantly in many tissues. This enzyme is not a

flavoprotein dependent enzyme; but requires NAD as coenzyme. In this case

the deamination is reversible (difference from the deamination by L-amino

acid oxidases). It brings the oxidation of glutamic acid to α-keto-glutaric acid

(α-oxoglutaric acid).

COOH COOH | gluamate | CH2 dehydrogenase CH2

| + NAD+ | + NADH + H+ CH2 CH2 | | oxidative CH.NH2 C=NH chain | | H2O + NAD+ COOH COOH Glutamic acid Imino acid COOH COOH | | CH2 H2O CH2

| | + NH2 CH2 CH2 | | C=NH CO | | COOH COOH Imino acid α-ketoglutaric acid

A number of agents dissociate the enzyme into sub-units (probably four) and

inhibit its activity as a glutamate dehydrogenase. Agents acting in this way

include (in the presence of NADH) ATP and GTP, oestrogens, androgens,

progesterone, and thyroxine; ADP, NAD+ and NADP+ act in the opposite

direction.

Since the above reaction is reversible, it functions both in amino acid

catabolism and biosynthesis. The latter (biosynthetic) function is of particular

importance in plants and bacteria, which can synthesise large amounts of

amino acids from glucose (source of α-keto acid) and ammonia.

243

Glycine is another amino acid which is acted upon by a specific enzyme

glycine oxidase.

glycine oxidase H2N.CH2.COOH + 1/2O2 CHO.COOH + NH3 Glycine glyoxalic acid

Lastly since the amino acids serine and threonine have one hydroxyl group

in their molecules, they are deaminated non-oxidatively by enzymes known

as dehydrases

CH2OH CH2 CH2

| serine | H2O | CH.NH2 C=NH C=O + NH3 | dehydrase | | COOH COOH COOH Serine Imino acid Pyruvic acid

2.Transamination : This is the most important mechanism for the

conversion of an amino acid to a keto acid and involves the transference

of an amino group from a donor amino acid to a recipient keto acid under

the influence of a transaminase or aminotransferase.

R1 R2 R1 R2

| | | | CH.NH2 + CO C O + CH.NH2 | | | | COOH COOH COOH COOH I II III IV

The donor amino acid thus becomes a keto acid and recipient keto acid

becomes an amino acid, the coenzyme required for this reaction is

pyridoxal phosphate.

Pyridoxal phosphate reacts with the amino acid to form a Schiff‟s base

type complex which then yields the keto acid and pyridoxalamine

phosphate. The latter now reacts with a second keto acid to form a

Schiff‟s base complex which again decomposes to produce an amino acid

and pyridoxal phosphate.

244

However, there are certain limitations to the general transamination

reaction. Although most of the amino acids may act as donor (I), the

reciepient keto acid (II) may only be either α-keto-glutaric acid or

oxaloacetic acid or Pyruvic acid. The amino acid (IV) formed from these

three keto acids are glutamic acid, aspartic acid and alanine, respectively.

Thus on the whole there are mainly three types of transaminases; out of

which that involving α-ketoglutaric acid as the recipient keto acid (II) is the

most important type.

COOH COOH COOH COOH | | | | CH2 CH2 CH2 CH2 | + | | + | CH.NH2 CH2 CO CH2

| | | | COOH CO COOH CH.NH2 | | COOH COOH Aspartic acid α-ketoglutaric acid Oxaloacetic acid Glutamic acid

The reaction is catalysed by the aspartate aminotransferase also known as

glutamate-oxaloacetate transaminase (GOT). The normal value of this

transaminase in the blood serum (SGOT) is 30-40 units/100ml. When there

is much damage to the cardiac and hepatic tissues, of course, with high

concentration in heart and liver tissues.

Most of the amino acids (but not all) undergo transamination except lysine,

threonine and the cyclic amino acids, proline and Hydroxyproline. Moreover,

transaminations involving the β , γ , or δ-amino acids, aldehydo-acids, and

even D-amino acids (in bacteria) are also known.

245

CH.NH2 COOH CHO COOH | | | | CH2 CH2 CH2 CH2 | | | | CH2 + CH2 CH2 + CH2 | | | | CH.NH2 CO CH.NH2 CH.NH2 | | | | COOH COOH COOH COOH L-ornithine α-ketoglutaric acid L-glutamate glutamic acid (a δ-amino acid) γ-semialdehyde

Since L-glutamate is the only amino acid in mammalian tissues which can

undergo oxidative deamination owing to the presence of highly active and

widely abundant L-glutamate dehydrogenase, all other amino acids are

converted to glutamic acid by transamination with α-ketoglutaric acid. The

glutamic acid then undergoes oxidative deamination to form α-ketoglutaric

acid and ammonia.

The process of amino acid catabolism by the combined action of an

aminotransferase (transaminase) and glutamate dehydrogenase may now be

summarized as below.

NH2 | R―CH―COOH α-ketoglutaric Amino acid acid NH3 Transaminase glutamate dehydrogenase Glutamic R―CO―COOH acid keto acid

overall catabolism (transdeamination) of amino acids

This process takes place mainly in the liver but occurs also in the kidneys. In

the liver, ammonia is converted into urea.

246

3.Transamidation: In this reaction, the amide group of glutamine is

transferred to a keto group. If the amide group is transferred to an α-keto

acid, an amino acid is formed ; while if the amide group is transferred to the

keto group of fructose, glucosamine is formed. Transamination and

transamidation serve in the interconversion of amino acids and synthesis of

the non-essential amino acids. Liver is the most active site for both of these

reactions.

Disposal of the Nitrogen. Since ammonia, constantly produced in the

tissues by the processes described above, is very toxic compound, it must

rapidly be removed from the circulation (detoxication). This function is

performed by the liver which removes ammonia rapidly from the circulation

by converting it to glutamate, glutamine, or urea. Liver performs this function

so rapidly that only traces of ammonia (10-20μg/100ml.) are present in the

blood. In the event of failure of hepatic function, concentration of ammonia

ion in blood increases and different tissues including brain are poisoned.

Other disadvantages of increased ammonia concentration is that it converts

α-keto acids to amino acids nd thus hinders Kreb‟s cycle ( a major source of

energy in the brain) reactions. The various paths for he fixation of ammonia,

obtained from amino acids, acids are : (i) synthetic pathways, (ii) glutamine

pathways, (iii) formation of urea, (iv) direct excreation, and (v) formation of

creatine and creatinine. Let us discuss them one by one.

1.Synthetic pathways. Ammonia may be used in the reductive amination of

α-keto acids, (derived from carbohydrate) to form new amino acids (reversal

of transdeamination reaction).

O NH2 || | R.C.COOH + NH3 R.CH.COOH

247

Ammonia may also be used for the synthesis of purines, pyrimidines and

porphyrins, although in these compounds ammonia is generally introduced in

the form of a carrier, such as glutaminate, aspartate, carbamyl phosphate,

and glycine, rather than in the free state.

2.Glutamine pathways (detoxication) : Free ammonia is a toxic substance

in cells and may lead to coma unless removed or detoxified. However, in the

extrarenal tissues it is converted into glutamine. The metabolic reaction of

ammonia leading to the formation of glutamine is catalysed by glutamine

synthetase, a mitochondrial enzyme present primarily in the brain and liver.

This reaction resembles somewhat with the synthesis of a peptide linkage,

and similarly requires a source of energy, i.e. ATP.

COOH CO NH2 | | CH2 CH2 | glutamine | CH2 + NH4 + ATP CH2 +ADP + H3PO4 | synthetase, | CH.NH2 Mg++ CH.NH2 | | COOH COOH L-Glutamic acid L-Glutamine The mechanism of this reaction presumably involves the intermediary

formation of γ-glutamyl phosphate and the subsequent exchange of the

phosphate group for the ― NH2 group. Glutamine acts as NH2 donor in

general metabolism, e.g. in the synthesis of purines and of glucosamine.

Glutamine is an important form for transporting ammonia in the organism

because in this form ammonia is no longer toxic. Actually the glutamine

travels from the various tissues through the blood to the kidneys, where it is

hydrolysed by glutaminase to glutamic acid and ammonia.

CONH2 COOH | | CH2 CH2

| glutaminase |

248

CH2 CH2 + NH2 | (+ H2O) | CH.NH2 CH.NH2 | | COOH COOH L-Glutamine L-Glutamic acid

The ammonia which is thus liberated accounts for about 60% of the urine

ammonia. An analogous reaction is the formation and hydrolysis of another

acid amide (asparagine) catalysed respectively by the enzymes asparagines

synthetase and asparaginase. Glutaminase and asparaginase have been

employed as anti-tumour agents since certain tumours exhibit abnormally

high requirements for glutamine and asparagines.

3.Direct excretion: usually deamination of amino acids occurs in ectrarenal

tissues where the ammonia is immediately channeled into certain metabolic

pathways which bind it. In case the removal of amino group from the amino

acid (deamination) occurs in kidney in the absence of immediate

physiological requirements for synthetic purposes, the liberated ammonia

may be excreted directly into the urine. This source of urinary ammonia

accounts to about 40% of the total urinary ammonia (60% of urinary

ammonia is derived by the hydrolysis of glutamine in kidney). It is important

to note in this respect that the direct excreation of ammonia as ammonium

salts is very less (although it occurs in states of metabolic acidosis), the vast

majority of ammonia is excreted as urea.

4. Formation of Urea: The urinary area constitutes about 95% of the

excreted nitrogen. On an average, about 30 gm. of urea is excreted per 24

hours. It has conclusively been proved by experiments in animals that the

formation of urea occurs only in the liver. From liver it is released into the

blood, and then cleared by the kidney .The conversion of ammonia to urea in

the liver is not a simple combination of ammonia with carbon dioxide and

249

water to form ammonium carbonate for direct transformation to urea. Most

probably it occurs by way of the ornithine cycle, proposed by kreb. The

ornithine cycle is a cyclic process which, like the tricarboxylic acid cycle, can

be considered to start with a carrier molecule, the ornithine (amino acid).

Before the actual arnithine cycle take place, ammonia (derived by

deamination of amino acids) and carbon dioxide (derived from kreb cycle)

combine with the aid of ATP to form carbamyl phosphate (amidophosphate).

Since two molecules of ATP are required, it has been suggested that the

reaction occurs in two steps. In the first step carbon dioxide is activated with

the consumption of 1 mole of ATP in presence of Mg++ and N-acetyl

glutamate as a cofactor; the ―COOH group is bound presumably to the N

atom. In the second step active carbon dioxide unites with an ammonium ion,

with the aid of another ATP to form carbamyl phosphate. This step is

catalysed by the enzyme carbamyl phosphate synthetase present in liver

mitochondria.

O Mg++ CO2 + ATP C + ADP OP Activated carbon Dioxide

O Mg++ O C +NH3 + ATP H2N―C + ADP + Pi OP OP

In bacteria, glutamine rather than ammonia serves as a substrate for

carbamyl phosphate synthesis. The reaction is catalysed by the enzyme

carbamate kinase. After the formation of carbamyl phosphate, the proper

ornithine cycle begins in which the former compound reacts with the δ-amino

250

group of orthinine to form citrulline in the presence of ornithine carbamyl

transferase.

Citrulline then condenses with the amino group of aspartate to form

arginosuccinate. The reaction requires ATP and is catalysed by the

arginosuccinate synthetase. Arginosuccinate is cleaved reversibly to

fumarate and arginine by the enzyme argnosuccinase. Finally, arginine is

cleaved by the well-known enzyme arginase into urea and ornithine. This

completes the cycle; and orthinine molecule accepts another molecule of

carbomyl phosphate to repeat the cycle.

In summary, two moles of ammonia (from glutamate and aspartate) join with

one mole of CO2 to give one mole of urea. In the process, 3 moles of ATP

are consumed and thus the formation of urea, from the energy view point, is

a luxury in which the cell apparently indulges in order to escape the

deleterious effect of high concentration of free ammonia.

The fact that urea is synthesised via the ornithine-arginine cycle is proved

with the aid of isotopes. With the help of labeled carbon it has been proved

that the carbon of urea is derived from cabon dioxide. Similarly, when

ammonia or amino acids containing labeled nitrogen (N15) are fed to

animals, the labeled nitrogen is found in the urea of the urine and in the

arginine of the

tissue proteins. Of the four nitrogen atoms in arginine only the two in the

amidine group are involved in urea formation, while the other two remain in

the amino acids of the cycle.

Urea is a highly diffusible substance and is found in almost all the fluids of

the body. The concentration of urea in blood is usually between 20-35

mg./100ml. and actually changes with the nature of diet. It is high in

individuals taking high protein diets and vice versa. Blood urea level may

251

increase in cases of early and terminal nephritis, in renal damages, renal

disfunctioning, such as inmercury poisoning and double polycystic kidney, in

cardiac failure. On the other hand, lower concentration of urea in blood is

found in hepatic damage and nonhemorrhagic nephritis with edema. Urea is

mainly excreted by the kidney.

5.Creatine and creatinine: Creatinine (the anhydride of creatine) derived

from creatine, is a significant excreatory from of amino acid nitrogen.

Aconstant amount (related to muscle mass) of creatinine is excreated daily.

Formation of these compounds is discussed elsewhere because these are

formed only from three (glycine, arginine, and methionine) rather than the

entire group of amino acids.

(Glycine, Arginine, Thionine)

ATP ADP CH3 ―N―CH2―COOH CH3 ―N―CH2―COOH | | C=NH C=NH | | NH2 NH ~PO3H2 CREATINE PHOSPHOCREATINE CH3 ―N―CH2

H3PO4

H2O

C = O

HN= C ―NH CREATININE

Disposal of carbon skeleton: As we have already seen, amino acids yield

keto acids by remova of the amino group as ammonia. The fate of these keto

acids may be either of the following.

252

1.Synthetic pathway: Like the disposal of nitrogen, the α-keto acids

(resulting from deamination) also may be reductively aminated by reversal of

the transdeamination mechanism, thus reforming the original amino acids.

Like the deamination, this process is also continuous and the net change is

determined by physiological requirements. Certain fragments of the carbon

skeletons are also used for special synthesis which are described in the

metabolism of individual amino acids.

2.Glucogenic pathways: The carbon skeleton of most of the amino acids

are convertible to carbohydrates (gluconeogenesis from protein). Such amino

acids are known as glucogenic or antiketogenic amino acids. A few amino

acids which are involved in carbohydrate metabolism directly, are shown

below,

Glucose Pyruvate Oxaloacetate α- Ketoglutarate Alanine Aspartate Glutamate

The various glucogenic amino acids are given in the table given below.

Table of amino acids according to the fate of their carbon skeleton.

Glycogen forming

(Glycogenic)

amino acids

Fat forming

(Ketogenic) amino

acids

Both glycogen and fat

forming ( glycogenic as

well as Ketogenic)

amino acids

Alanine, Hydroxyproline,

Arginine, Methionine,

Aspartic acid, Proline

Cystine, Cysteine, Serine,

Glutamic acid, Threonine

Leucine Isoleucine

Lysine

Phenylalanine

Tyrosine

Tryptophan

253

Glycine, Valine, Histidine

The keto acids obtained from these amino acids may also directly enter the

tricarboxylic acid cycle and thus oxidized ultimately to CO2 and H2O. For

example, Pyruvic acid obtained by the deamination of alanine is oxidized to

CO2 and H2O via TCA cycle.

3. Ketogenic Pathway: The α-keto acid (isovaleryl formic acid) obtained

from the deamination of leucine, on its way of oxidation to CO2 and H2O

passes through the stage of acetoacetic acid and thus forms ketone bodies

instead of glucose. Such amino acids are known as ketogenic amino acids.

4. Glucogenic as well as ketogenic pathways: The keto acids obtained

from certain amino acids may enter the above mentioned glucogenic as well

as ketogenic pathways, i.e., they can give rise to both glucose and ketone

bodies. Such amino acids include isoleucine, lysine, phenylalanine, tyrosine

and tryptophan.

Thus on the whole, each amino acid in the form of its keto acid is convertible

either to carbohydrate (13 amino acids), fat (1 amino acid), or both (5 amino

acids);see the above table. This fact supports the concept of the

interconvertibility of fat, carbohydrate, and protein carbons which is also

confirmed by isotopically labeled amino acids.

5. Other pathways : Certain amino acids traverse metabolic pathways which

do not correspond with either of the above pathways. These routes are highly

individual, and are discissed in the appropriate sections.

Disposal of Sulphur: Sulphur is an important constituent of the body. It is

derived largely from proteins having cystine and methionine as amino acids.

In the body, the essential amino acid methionine may be converted into non-

essential amino acid cystine but not vice versa. Most of the Sulphur of these

254

amino acids is eventually oxidised to sulphate, which is excreted in the urine

mainly as inorganic sulphate with a small fraction as organic or ethereal

sulphate. Sulphur present in these sulphates is known as oxidized Sulphur.

The amount of oxidized Sulphur in the urine varies with the protein intake.

However, all of the Sulphur is not oxidized in the body. A constant amount,

known as neutral Sulphur, is always found in the urine in the unoxidized

form. Compounds containing neutral Sulphur of the urine in the unoxidized

form. Compounds containing neutral Sulphur of the urine are cystine, methyl

mercaptan, ethyl sulphide, thiocyanates, and taurine derivatives. In a certain

inborn error of metabolism, the ability of kidney to reabsorb cystine is

decreased and large amounts of cystine may be excreted in the urine

(cistinuria). Under certain conditions cystine may form deposits in the kidney

with serious consequences.

Energetics of Amino acid Oxidation: Due to the diverse catabolic

pathways and the multiplicity of the amino acids, it is difficult to form broad

generalization about the energetics of the process. However, if it is assumed

that most of the nitrogen derived from amino acids is excreted in the form of

urea, and that most of the keto acids are oxidized to CO2 ( HCO3 – at

physiological pH) and water through the kreb cycle, then it is possible to

arrive at a definite conclusion about the energetics of oxidation of a typical

amino acid. Let us discuss the energetics of glutamate oxidation since its

catabolic pathway is in accord with the foregoing assumptions.

The overall oxidation of one mole of glutamate yields bicarbonate, water and

half a mole of urea with the liberation of 490 kcal of free energy. The extent

of conservation of this energy in the from of ATP can be calculated as below.

1. Transdeamination of glutamate to α-ketoglutarate and ammonia yields a

reduced DPN or TPN which corresponds with 3 moles of ATP.

255

2. Since concersion of ammonia to urea is an endergonic process, and

requires the expenditure of 3 moles of ATP per mole of urea, the half mole of

urea formed during glutamate catabolism utilizes

1.5 moles of ATP.

3. Since the path of ketoglutarate to CO2 and water through kreb cycle

involves the oxidation of ketoglutarate to succinate (yielding 4 moles of

ATP), succinate to fumarate (yielding 2 moles of ATP), and malate to

oxaloacetate (yielding 3 moles of ATP), a total of 9 moles of ATP are

produced from this part of the cycle. The pyruvate, obtained by

decarboxylation of the oxaloacetate, then undergoes its usual oxidation to

CO2 and H2O and yields 15 moles of ATP. Thus on the whole 9+15=24

moles of ATP are produced from the complete oxidation of the carbon

skeleton.

By combining the above three steps it is clear that complete catabolism of

glutamate molecule yields a net amount of 3+24-1.5=25.5 moles of ATP.

Assuming an average energy of one ATP mole as 7.5 kcal; 7.5 x 25.5 = 191

kcal ( or 191x100/490 = 39%) of free energy is conserved in the complete

oxidation of glutamate.

1.6 CHEMISTRY OF OXYTOCIN HORMONE & TRYPTOPHANE

HARMONE.

1.6:1 Introduction: A hormone is commonly defined as a chemical

substance which is produced in one part of the body, enters the circulation

and is carried through the blood stream to distant organs or tissues (except

local hormones which function at the site of their production) to modify their

structure and function. These distant organs or tissues on which hormones

act are called as target cells or target organs. Like enzymes they act as

256

catalysts and are required only in very small amounts However, they differ

from enzymes in the following respects.

I. They are formed in one organ and perform functions in other

organ.

II. Before being utilized, these are always secreted into the blood.

III. Chemically, they may be proteins, polypeptides, single amino

acids, and steroids.

Although most of the hormones are produced by specialized cells known as

endocrine glands, ductless glands or glands of internal secretion, some are

also secreted by other organs, viz., secretion and cholecystokinin from GIT,

noradrenaline and acetylcholine from nerve-endings and erythropoietin and

rennin from kidney. Moreover, it is important to note that although the

term٬٬hormone‟‟ implies an ٬٬exiting‟‟ influence, certain hormones are now

known to exert a depressing effect on certain of their target tissues.

Mode of Action of Harmones: Hormone are found to act on three sites,

namely cell membrane, intracellular enzyme systems and the cell nucleus.

Chemical Nature of Hormones: Chemically, hormones are amino acid

derivatives, polypeptides or proteins, and steroids in nature. Thus chemically

there are three distinct types of hormones.

1.Steroid hormones: They contain a stetiod nucleus

(cyclopentenophenanthrene) in their molecules. Male sex hormones

(androgens), Female sex hormones (estrogens and gestogens) and

hormones of the adrenal cortex (adrenocorticoids) belong to this group.

2. Amino acid derivatives: A few of the hormones are amino acid

derivatives. Thyroxine, adrenaline and noradrenaline fall in this category.

3.Polypeptide and protein hormones: Hormones of this group are

proteinous in nature. This major group includes insulin, glucagons, oxytocin,

257

vasopressin, parathormone, hormones from the anterior lobe of the pituitary

body, and hormones from the GIT.

Posterior Lobe (Neurohypophyseal) Hormones: The posterior lobe of the

pituitary secretes mainly two hormones: vasopressin (pitressin) having the

pressor and antidiuretic effects, and oxytocin (pitocin) having the oxytoxic

effect. Although these two hormones differ greatly in physiological action,

they are very similar in chemical structure. Both of them are octapeptides

with one disulphide bridge. Oxytocin contains isoleucine in position 3, in

position 8 oxytocin contains leucine.

1 2 H.Cys------Tyr

| | S 3Ile

| | S 4Glu (NH2) | 6 5 | Cys――Asp(NH2)

| 7 8| Pro――Leu―Gly―NH2

Structure of Oxytocin Harmone

In the non-mammalian vertebrates (e.g. frog, other amphibians, reptiles,and

fishes),

These hormones and replaced by vasotocin, which has relatively weak

vasopressor and oxytoxic activity.

Oxytocin acts on the smooth muscles of the uterus and enhances

contraction. It undoubtedly plays a major role during parturition. It also

causes contraction of the smooth muscles of the lactating mammary gland,

causing ejection of milk. Oxytocin secretion is increased by suckling; it has

been suggested that it stimulates release of prolactin. It also stimulates

contraction of the gallbladder, intestine, urinary bladder and ureters. The

secretion of oxytocin is controlled by hypothalamus.

258

1.6:2 Tryptophan. (α-Amino-β-indolepropionic acid). It is the only amino

acid containing the indole ring. It is an essential amino acid. Tryptophan is

required in the dite for growth and for the maintenance of nitrogen balance in

the adult human. In rats the appetite fails and the serum albumin and globulin

fall markedly, if the tryptophan is ommited from the diet. Haemoglobin also

diminishes and the animals develop cataracts.

In addition to its importance as a constituent of proteins, this amino acid is

also a source of several compounds of importance to cellular metabolism.

Tryptophan replaces nictotinic acid as a dietary constituent for growth

maintenance since it is converted to nicotinic acid via pathway of kinurenine.

It is also a precursor of serotonin (5-HT), a substance which is present in a

number of tissues e.g. blood,GIT, GHS. Serotonin is a powerful

vasoconstrictor, smooth muscles stimulator and probably a neurotransmitter.

The plant growth hormone indoleacetic acid (auxin) also originates from

tryptophan.

The D- and L-isomers of tryptophan both can maintain nutrition and growth

equally well, indicating that the unnatural D-isomers is converted to L-isomer

via the common indole pyruvic acid. Hence indole pyruvic acid,

corresponding keto acid of tryptophan, hydroxyl acid (indole lactic acid), etc.

can replace the tryptophan in the diet Metbolic breakdown. It is metabolized

through several pathways which vary somewhat in different species of

animals. The important pathways is discussed below one by one.

(A) Kynurenine or Nicotinamide Pathway. This is the main pathway for the

metabolic breakdown of tryptophan. By this pathway, tryptophan is converted

into nicotinic acid (a vitamin), NAD+ and NDA+P.It appears contradictory to

the statement that a vitamin is formed by an organism, but nicotinamide

deficiency can indeed be demonstrated only with a tryptophan-poor diet and

259

the concurrent deficiency of vitamin B6. Tryptophan largely replaces the

vitamin nicotinamide even in man.

The catabolism of tryptophan seems to have many steps in common with the

biosynthesis of nicotinamide. Initially the tryptophan molecule is attacked by

the enzyme tryptophan pyrrolase (oxygenase) which opens up the indole

ring. As in other cases of oxidative ring opening, the double bond is split and

each new end receives one oxygen atom to form N-formylkynurenine. The

latter compound is then hydrolyzed to formate and kynurenine; the hydolysis

is catalysed by kynurenine formylase of mammalian liver.

Kynurenine may be deaminated by transamination of the amino group of the

side chain to the diketo derivative whioch undergoes spontaneous cyclisation

by losing water to form kynurenic acid. Kynurenic acid is a by-product of

kynuresine; it is not formed in the main pathway of tryptophan breakdown.

Kynurenine is oxidized by atmospheric oxygen to yield 3-hydrxy kynurenine

(an NADPH-catalysed reaction). 3-hydrxy kynurenine is then cleaved to yield

3-hydroxyanthranilate and alanine; the reaction is catalysed by the enzyme

kynureninase which requires pyridoxal phosphate (vitamin B6) as coenzyme.

3-hydrxyanthranilate is subjected to another oxidative ring opening adjacent

to the hydroxyl group to form 2-acroleyl-3-amino-fumarate.

However, in case of vitamin B6 deficiency, kynurenine derivatives are not

cleaved and thus reach various extrahepatic tissues (e.g. kidney) where they

are converted to xanthurenic acid (an abnormal metabolic of tryptophan)

which has been identified in human urine.

2-Acroleyl 3-aminopfumarate is very unstable and may undergo catabolism

mainly in two important ways to form different products.

Check your progress- 2

1. Write your answer in the space given below.

260

2. Check your answer with the one at the end of the unit.

Fill in the blanks:-

1. An example of α helix of protein---------------------------.

2. An example of β sheets of protein--------------------------.

3. ----------------------- is an example of quarternary structure of protein.

4. Hormone oxytoxin is ------------------------------------------.

1.7 LET’S SUM UP

Proteins are regarded as the most important constituents of all living

organisms as they play important role in their life. The important functions of

proteins are (a) They form the structural framework of the cell, (b) Maintain

osmotic balance, (c) catalyse biochemical reactions, (d) regulate metabolism,

(e) help in stroge of some elements, (f) act as oxygen carriers, (g) from the

colloidal system in protoplasm, (h) transport lipids as lipoproteins, and (i) act

as storage proteins ( proteinoplasts). They are polymers of amino acids.

The amino acids are derivatives of carboxylic acids in which a hydrogen

atom in a α-carbon chain is replaced by an amino group (-NH2). They are

represented by a general formula shown below.

H | α R―C―COOH | NH2

( where R-represents a great variety of structures).

Proteins, on hydrolysis yield a mixture of α-amino acids.They are produced

from natural proteins by the action of enzymes, acids or alkalies.

Primary Structure

261

The sequential arrangement of the various amino acids in a protein

(Polypeptide chain) through the peptide bonds is known as the primary

structure. Each protein molecule consists of one or more polypeptide chains

in which the aminoacids are linked by peptide linkages. Myoglobin, a protein,

consists of only single polypeptide chain, whereas hemoglobin molecule

consists of four polypeptide chains. The covalent bonds and disulphide

(―S―S―) bonds are again characteristics of the primary structure of

proteins. The disulphide bond is generally established between cysteine

residues, as in insulin and ribonucleases.If the protein has only one

polypeptide chain, it can have only one free α-amino group(-NH2 terminal)

and one free carboxyl (-C-terminal) group. In the determination of primary

structure of protein, it is essential to know what amino acids are a N-terminal

and C-terminal ends. The free α-amino group can react with the

reagents like dinitrofluorobenzenes to give a dinitrophenyl (DNP)

derivative which on hydrolysis yields the yellow coloured DNP–amino acids.

These can be isolated chromatographically.

Secondary Structure of Protein

The covalent backbone of a polypeptide chain is formally single-bonded. We

would therefore expect the backbone of a polypeptide chain to have an

infinite number of possible conformations and the conformation of any given

polypeptide to undergo constant change because of thermal motion.

However, it is now known that the polypeptide chain of a protein has only one

conformation (or a very few) under normal biological conditions of

temperature and pH. This native conformation, which confers biological

activity, is sufficiently stable so that the protein can be isolated and retained

in its native state. This fact therefore impiles that the single bonds in the

backbone of native proteins cannot rotate freely. When the long polypeptide

262

chains in a protein undergo folding, they form the secondarystructure or

helical structure. The secondary structure is determined by hydrogen

bonding between the components of the peptide chain itself. The hydrogen

bonds can occur either within one polypeptide chain or between different

polypeptide chains of the protein molecule.Thus, the secondary structure of

proteins is represented by helical structures which are ultimately formed by

the hydrogen bonding between the chains or chain.

Fine Structure of Proteins

α-Structure: Fine structure of proteins was discovered by Pauling and Corey

(1951) of California Institute of Technology and were awarded Nobel Prize in

chemistry for this discovery. They proposed the α-helical conformation of

protein based on theoretical grounds.

β-Structure : Asbury and Street (1933) proposed β-Structure of proteins

which was later modified by Pauling and Corey. The β-Structure is

represented by parallel zig-zag polypeptide chain which form a pleated

sheet-like structure. The hydrogen bonds are formed between NH and C=O

groups on the neighbouring chains which stabilize the β-Structure of

proteins. The side chain attached to the amino acid residues lies above and

below the hydrogen bonded sheets. This structure is a stable arrangement

where side chains are small and do not cause distortion of the pleated

structure. In fibroin, the chains run anti-parallel to each other, i.e the free

amino (or carboxyl) groups are at opposite ends of neighbouring chains. β-

Structure is found in milk and keratin.

Tertiary Structure

Very few protein molecules exist as a simple α-helix. Further degrees of

folding or coiling of polypeptide chains in α-helix give a complex three-

dimensional structure (tertiary structure) which often contains helical and

263

non-helical regions. Folding of the α-helix occurs where the amino acid

proline has an imino group instead of an amino group which causes

unstability in the α-helix by producing hindrance in the regular internal

hydrogen bonding. Three main types of bonds, ionic, hydrogen and

hydrophobic, are responsible for the formation of the tertiary structure of a

protein. Dipole-dipole interaction and disulphide linkages are also

responsible for the formation of tertiary structure. Tertiary structure of

proteins is probably thermodynamically the most stable and is of most stable

and is of much importance, because the enzymatic properties of a protein

depend on it.

Quaternary Structure

This defines the degree of polymerization of a protein unit. The quaternary

structure is exhibited by haemoglobin molecule which was determined by

Perutz and coworkers (1960). They showed that this protein undergoes

further organization, being made up of 4-polypeptide chains. This further

organization is known as the quaternary structure. The chains undergo

secondary folding, two of the structures consisting of α-chain and other two

of β-chain. All 4-chains fit together in a compact tetrahedral arrangement to

form a complete haemoglobin molecule. The forces maintaining the

quaternary structure are similar to those involved in tertiary structure, but the

association of the sub-units, in general is more flexible.

264

1.8 CHECK YOUR PROGRESS:- THE KEY

Check your progress- 1 :- The Key

1.

a) refer point:-α-helix

b) refer point:-β- Conformation

c) refer point;- Heamoglobin

2. Name of scientist, step to determine terminal residue end, terminal

residue reagent used , etc.

Check your progress- 2 :- The Key

1) horns, nails

2) fibres spun by spiders & silkworms.

3) Haemoglobin.

4) A polypeptide hormone

1.9 Assignment/ Activity

1. Draw parallel & antiparallel β sheets of keratins.

2. Write a detail note of primary, secondary , tertiary & quaternary

structure of proteins.

2.0 REFERENCES.

1. Stryer lubert , Biochemistry.

265

2. Lehninger, Biochemistry , Kalyani Publication

3. Jain, J-L , Biochemistry , S.Chand Publication.

4. Agarwal‟s , A text book of biochemistry.

Sterioisomerism ↓

-----------------------------------------------------------------------------

266

↓ ↓ Geometrical Optical This occurs due to occurrence of two ↓ different atoms at two valancies of carbon ---------------------------------- ↓ ↓ ↓ ------------------------- Conformational Configurational ↓ ↓ ↓ ↓ Cis trans Interconvertible Arrangement that Arrangement can‟t be changed.

267

UNIT V

Nucleic acids

5.1 Introduction

5.2 Objectives

5.3 The chemical basis of heredity

5.4 Purine and pyrimidine bases of nucleic acids.

5.5 Double helical model of DNA(Watson & Crick’s model) and

forces responsible for holding it.

5.6 Chemical and enzymatic hydrolysis of nucleic acids.

5.7 Structure of RNA

5.8 Replication of DNA

5.9 Transcription

5.10 Translation and genetic code

5.11 Chemical synthesis of mono and trinucleosides

5.12 Let us sum up

5.13 Check your progress key

5.14 Assignment.

5.15 References

268

3.1 INTRODUCTION

The nucleic acids are of considerable importance in biological systems.Two types of

nucleic acids are found in the cells of all living organisms. These are:

1. Deoxyribonucleic acid - DNA

2. Ribonucleic acid - RNA

The nucleic acid was first isolated by Friedrich Miescher in 1868 from the nuclei of

pus cells and was named nuclein. At that time its biological significance was barely

understood. The name nucleic acid was given to it after knowing its acidic property.

They are of two types; (1) Ribose nucleic acid, and(2) Deoxyribose nucleic acid .

The basic chemical subunits of the nucleic acids are nucleotides. The nucleotides are

made up of three components: (i) A heterocyclic ring containing nitrogen, known as

a nitrogenous base, (ii) a five carbon pentose sugar, and (iii) A phosphate group.

The bases found in nucleic acid are of two kinds- purines and pyrimidines.Adenine

and guanine are purine and cytosine, uracil and thymine are pyrimidine bases. The

nucleotides found in nucleic acids are much fewer in number than the α-amino acids.

DNA is found in almost all the cells as a major component of chromosomes of the

nucleus. In 1962 the presence of chloroplast DNA was reported by Ris and Plant

from Chlamydomonas. Segments of DNA as long as 150μ have been reported from

chloroplasts by Woodcock and Fernandez-Morgan (1968). In 1963 M.M.K. Nass and

S.Nass reported the presence of DNA from mitochondria. Certain viruses, including

many of the bacterial viruses or bacteriophages, are DNA-protein particles. Mostly

the plant viruses are RNA-protein particles.

Ribose nucleic acid (RNA) is also of common occurrence in plants as well as

animals. It is of three types- (i) ribosomal RNA (r-RNA); (ii) soluble RNA or

transfer RNA (t-RNA) and (iii) messenger RNA (m-RNA). Ribosomal-RNA is

found in small sub-cellular particles, the ribosomes. RNAs with sendimentation

Coefficient value, 5S, 16S and 23S have been reported from 70S ribosomes, while

269

18S, 28S, 5.8S and 5S r-RNAs have been reported from 80S ribosomes. T-RNA is

found in free from in the cytoplasm. M-RNA is found in small quantities in

association with ribosomes.

3.2 OBJECTIVES

This unit emphasizes on understanding the;-

1. Structure of purines, pyrimidines, & Nucleic acid.

2. Structural and conformational details of DNA and RNAs.

3. The process of DNA replication & protein synthesis

4. Basic requirement to move a step towards molecular and genetic studies.

3.3 THE CHEMICAL BASIS OF HEREDITY

DNAs are present mainly in the nucleus (in chromosomes) of the cell so they are the

carrier of gnetic information because the DNA molecule can produce a copy of itself

each going to one cell i.e. the parent DNA molecule gives rise to two identical

daughter molecules each going to one cell and thus each daughter cell receives

exactly the same complement of DNA (both qualitatively and quantitatively as the

parent cell.

DNA as the bearer of genetic information in the cell is strongly supported by the

Watson-Crick structure for this compound which explains beautifully the

phenomenon of replication (and hence genetic continuity) of DNA.

This phenomenon of DNA replication can be explained as below: the double helix of

DNA separates into two strands: the individual strands combine in sequence with

their complementary free nucleotides (present in nuclear sap) through specific

hydrogen bonding (Adenine….Thymine, Guanine…Cytisine) and now phosphate

ester linkages are formed between two nucleotides by the enzyme catalase.

270

Thus DNA in animals, plants, bacterial cells, and some viruses maintain the genetic

continuity. The direct evidence in favour of the genetic role of DNA is derived from

the process of bacterial transformation. If an extract of the strain of pneumococcus,

possessing capsules having specific polysaccharides, is added to the culture of strain

of pneumococcus not having the above mentained capsules, the latter is transformed

to the former. The active agent (transforming factor) is a DNA molecule which

endows the bacterial cell with the capacity for synthesizing an enzyme or enzyme

system not present previously in non-encapsulated strain. This enzyme in turn

catayzes the formation of the specific capsular polysaccharide.

DEOXYRIBONUCLEIC ACID - DNA

Occurrence

271

DNA is found in the cells of all living organisms except plant viruses, where RNA

forms the genetic material and DNA is absent. In bacteriophages and viruses there is

a single molecule of DNA, which remains coiled and is enclosed in the protein coat.

In bacteria, mitochondria and plastids of eukaryotic cells DNA is circular and lies

naked in the cytoplasm. In the nuclei of eukaryotic cells DNA occurs in the form

long spirally coiled and unbranched threads. The number of DNA molecules is

equivalent to the number of chromosomes per cell. In them DNA is found in

combination with proteins forming nucleoproteins or the chromatin material and is

enclosed in the nucleus.

3.4 PURINE AND PYRIMIDINE BASES OF NUCLEIC ACID.

Chemical composition

The Chemical analysis has indicated that DNA is composed of three different types

of compounds:

4. Sugar Molecule represented by a pentose sugar, the deoxyribose or 2‘-

deoxyribose.

5. Phosphoric Acid.

6. Nitrogenous Bases: These are nitrogen containing organic ring compounds.

These are of the following four types:

I. Adenine represented by – A

II. Thymine represented by – T

III. Cytosine represented by – C

IV. Guanine represented by – G

272

DEOXYRIBONUCLEIC ACID or DNA

These four nitrogenous bases are separated into two categories:

(c) Purines: These are two-ringed nitrogen compounds. Adenine and guanine are

the two purines found in DNA. Their structural formulae are represented in

fig.2.

(d) Pyrimidines: These are formed of one ring only and include cytosine and

thymine. Chemical analysis of DNA further reveals three fundamental

features described by Chargaff and is called Chargaff’s base ratio.

Molecular Structure

273

The constituents of DNA were isolated quite early but how these are arranged so as

to carry out their cytological and genetical activities was not known for long. DNA is

a long chain polymer was clearly understood in late 1930s. However, in 1953, D.S.

Watson and F.H.C. Crick presented a working model of DNA. This model illustrates

not only its chemical structure but also the mechanism by which it duplicates itself.

1.Nucleosides

A nitrogenous base with a molecule of deoxyribose (without phosphate group) is

known as nucleoside. The nitrogenous base is attached to first carbon atom C-1 of

deoxyribose N-glycosidic bond. In all, there are four nucleosides in a DNA

molecule. These are:

1. Adenosine – Adenine + Deoxyribose

2. Guanosine – Guanine + Deoxyribose

3. Cytidine – Cytosine + Deoxyribose

4. Thymidine – Thymine + Deoxyribose

2. Nucleotides ( The Monomers of DNA)

A nucleotide is formed of one molecule of deoxyribose, one molecule of phosphoric

acid and one of the four nitrogenous bases. Since there are four nitrogenous bases,

there are four type of nucleotides namely:

5. Deoxyadenylic acid -Adenine + Deoxiribose + Phosphoric acid

6. Deoxyguanylic acid -Guanine + Deoxiribose + Phosphoric acid

7. Deoxycytidylic acid -Cytosine + Deoxiribose + Phosphoric acid

8. Deoxythymidylic acid -Thymine + Deoxiribose + Phosphoric acid

1. Polynucleotide Chain (Linking of Nucleotides in a DNA Molecule)

DNA is a macromolecule formed by the linking of several thousand nucleotides.

These are called monomers or building blocks of DNA. In a nucleotide the

phosphate (phosphoric acid) molecule is attached to fifth carbon atom (C-5) of the

deoxyribose molecule through a phosphodiester linkage. The adjacent nucleotides

274

are connected together forming the sugar phosphate chain in which sugar and

phosphate molecule are arranged in alternate fashion. The phosphate molecule of a

nucleotide is joined to the third carbon atom of the deoxyribose. These are directed at

right angles to the long axis of the polynucleotide chain and are stacked one above

the other.

Marked Ends of Polynculeotide Chain: Each polynucleotide chain has

marked ends. Its top end has a sugar residue with free 5‘ carbon atom which is

not linked to another nucleotide. The triphosphate group is still attached to it.

This end is called the 5’ or 5’-P terminus. The other end of the chain ends in a

sugar residue with C-3 carbon atom not linked. It bears 3‘-OH group. This end

of polypeptide chain is called 3’ end or 3’-OH terminus. It means the

poplypeptide chains have direction and are marked as 3‘-5‘.

3.5 DOUBLE HELICAL MODEL OF DNA (WATSON & CRICK’S MODEL)

AND FORCES RESPONSIBLE FOR HOLDING IT.

.Watson and Crick’s Model of DNA

Watson and Crick suggested that in a DNA molecule ther are two such

polynucleotide chains arranged antiparallel or in opposite directions i.e., one

polynucleotide chain runs in 5‘-3‘ direction, the other in 3‘-5‘ direction. It means the

3‘end of one chain lies beside the 5‘ end of other. In such a structure the phosphate

groups of nucleotides in each polynucleotide chain or strand lie on the outside of the

deoxyribose and the nitrogenous bases are directed inward. The nitrogenous bases of

the two chains are linked through hydrogen bonds formed between oxygen and

nitrogen atoms of the adjacent bases. The unique feature of pairing between bases is:

1. Purine (adenine and guanine) pairs with pyrimidine (cytosine and

thymine), and

2. Adenine pairs with thymine and cytosine pairs with guanine.

There are definite reasons for such a specific pairing :

275

1. Such pairing forms a perfect match between hydrogen donor and hydrogen

acceptor sites on the two molecules. Adinine and thymine share two

hydrogen atoms, whereas cytosine and guanine are joined by three hydrogen

bonds.

2. Such a pairing is further supported by the occurrence of constant diameter of

DNA. In a limited area a two-ringed molecule (purine) joins a single-ringed

molecule maintaining a constant and roughly equal distance. A and G pair will

be rather too large to fit inside the helix and C and T would appear to be far

apart.

Due to this type of base pairing the two strands are complementary to each other. It

means if a chain has a region with a sequence of nitrogenous bases, thymine-

cytosine-adenine-cytosine-guanine, then the corresponding region in the

complementary chain will have the base sequence adenine-guanine-thymine-

guanine –cytosine.

DNA consists of two complementary chains twisted around each other forming a

right-handed helix. One turn of helix measures about 34 Å. It contains 10 pairs of

nucleotides placed at regular intervals of 3.4 Å. The diameter of the helix is roughly

20Å. A narrow helical groove and a wide helical groove run along the length of

DNA helix. The narrow groove is the distance between the paired molecules while

the wide groove is the space between successive turns when the pair is wound into a

helix.

In 1953, James Watson and Francis Crick deduced the three dimensional structure of

DNA and immediately inferred its mechanism of replication, Watson and Crick

analyzed X-ray diffraction photographs of DNA fibres taken by Rosalind Franklin

and Maurice Klilkins and derived a structural model that has proved to be essentially

correct.

The salient features of their model are:-

276

9. Two helical polynucleotide chains are coiled around common axis, the chains

run in the opposite directions.

10. The purine and pyrimidine bases are on the inside of the helix, where as the

phosphate and deoxyribose units are on the outside the planes of the bases are

perpendicular to the helix axis. The planes of the sugars are nearly at right

angles to those of the bases.

11. The diameter of the helix is 20 A0, adjacent bases are separated by 34 A

0 along

the helix and related by a rotation of 360, hence the helical structure repeats

after in residues on each chain, i.e., at interval of 34A0.

12. The two chains are held together by hydrogen bonds between the pairs of

bases adenine is always paired with thymine guanine is always paired width

cytosine.

13. The sequence of bases along a polynucleotide chain is not restricted in any

way, the precise sequence of bases carries the genetic information.

14. The ratio of A+G/C+T always equals to one

15. In every organism, the sequence of nucleotides in constant. The ratio of

A=T/G=C is also specific to organisms.

16. Each pitch of DNA has two major and two minor groves.

Fig.

DNA

Structure

The

most

importa

277

nt, aspect of the DNA double helix is the specificity of the pairing of the bases.

Watson and Crick deduced that adenine must pair with thymine and guanine with

cytosine.

The steric and hydrogen bonding factors restriction is imposed by the regular helical

nature of the sugar-phosphate backbone of each polynucleotide chain the glycosidic

bonds that are attached to a bonded pair of bases are always 10.85 A apart a pair of

pyrimidine base pair fits perfectly in this shape, in contrast, there is insufficient room

for two purines. There is more than enough space for two pyrimidine but they would

be too far apart to form hydrogen bonds Hence, one member of a base pair in a DNA

helix must always be a purine the other a pyrimidine, because of stric factors. The

base pairing is further restricted by hydrogen bonding requirements. The hydrogen

atoms in the purine and pyrimidine bases have well defined positions. Adenine can

not pair with cytosine because there would be two hydrogen near one of the bonding

positions and none at the other like wise guanine can‘t pair with thymine, where as

guanine forms three bonds with cytosine. The orientation and distance of those

hydrogen bonds are optimal for achieving strong interaction between the bases. The

base pairing scheme was strongly supported by the base compositions of DNA‘s

from different types. In 1950, Erwin chargaff found that the ratios of adenine to

thymine and guanine to cytosine were nearly 1 in all the samples studied.

278

Check your progress-1

1. Note :- Write your answer in the space given

2. Compare your answer with the one given at the end of the unit.

a. Name purines & Pyrimedines bases of DNA.

b. What does Chargaff rule says ?

c. Explain the structure of DNA.

----------------------------------------------------------------------------------------------

----------------------------------------------------------------------------------------------

-----------------------------------------------------------------------------------------------

-----------------------------------------------------------------------------------------------

-----------------------------------------------------------------------------------------------

-----------------------------------------------------------------------------------------------

-----------------------------------------------------------------------------------------------

-----------------------------------------------------------------------------------------------

-----------------------------------------------------------------------------------------------

-----------------------------------------------------------------------------------------------

-----------------------------------------------------------------------------------------------

-----------------------------------------------------------------------------------------------

-----------------------------------------------------------------------------------------------

-----------------------------------------------------------------------------------------------

-----------------------------------------------------------------------------------------------

-----------------------------------------------------------------------------------------------

-----------------------------------------------------------------------------------------------

-----------------------------------------------------------------------------------------------

-----------------------------------------------------------------------------------------------

-----------------------------------------------------------------------------------------------

279

------------------------------------------------------------------------------------------------

------------------------------------------------------------------------------------------------

------------------------------------------------------------------------------------------------

------------------------------------------------------------------------------------------------

------------------------------------------------------------------------------------------------

------------------------------------------------------------------------------------------------

------------------------------------------------------------------------------------------------

------------------------------------------------------------------------------------------------

------------------------------------------------------------------------------------------------

-------------------------------------------------------------------------------------------------

-------------------------------------------------------------------------------------------------

-------------------------------------------------------------------------------------------------

3.6 CHEMICAL AND ENZYMATIC HYDROLYSIS OF NUCLEIC ACIDS.

Hydrolysis or Degradation of RNA

Like DNA, RNA can also be hydrolysed under different conditions. The acid

hydrolysis yields the different components which actually form it. The various

components are free purines and pyrimidines, ribose sugar and phosphoric acid

280

Showing hydrolysis of DNA

3.7 STRUCTURE OF RNA

In all other organisms, where DNA is the hereditary material, different types of RNA

are nongenetic. In general, three types of RNAs have been distinguished:

1. Messenger RNA or nuclear RNA (mRNA)

2. Ribosomal RNA (rRNA)

3. Transfer RNA (t RNA)

Messenger RNA or Nuclear RNA

281

mRNA is synthesised inside the nucleus as a complementary strand to DNA and

carries genetic informations from chromocomal DNA to the cytoplasm for the

synthesis of proteins. For this reason only, it was named messenger RNA (mRNA)

by Jacob and monod in 1961. It has following characteristics.

(a) It is formed as a complementary strand to one of the two strands of a DNA.

(b)The thymine of DNA is substituted by uracil in mRNA. mRNA, therefore,

contains the same information as coded in that part of DNA.

(c)After synthesis it immediately diffuses out of the nucleus into the cytoplasm,

where it is deposited on certain number of ribosomes.

(d)Here mRNA acts as a template for protein synthesis.

(e)It has a short life span and withers away few translations.

Monocistronic mRNA molecule containing the codons of a single cistron, which

codes for one complete molecule of protein.

Polycistronic mRNA molecule containing the codons for more than one cistron

which may lie close together. This type of mRNA synthesises more than one protein

chains. For example, mRNA molecule which governs the metabolism of histidine

codes for the synthesis of ten specific enzymes.

Transfer RNA (tRNA)

The transfer RNA is a family of about 60 small sized ribonucleic acids which can

recognize the codons of mRNA and exhibit high affinity for 21 activated amino

acids, combine with them and carry them to the site of protein synthesis. tRNA

molecules have been variously termed as soluble RNA or supernatant RNA or

adaptor RNA. tRNA is about 10-15 percent of total weight of RNA of the cell. Its

molecules have following characteristics;

1. tRNA molecules are smallest, containing 75 to 85 nucleotides.

2. Its polynucleotide chain is bent in the middle and folded back on itself ( clover

leaf model) and the two arms coiled over one another.

282

3.The 3‘ end of the polynucleotide chain ends in CCA base sequence. This represents

site for the attachment of activated amino acid. The end of the chain terminates with

guanine base.

4.The band in chain of each tRNA molecule contains a definite sequence of three

nitrogenous bases which constitute the anticodon. It recognizes the codon on mRNA.

5. Four different regions or special sites can be recognized in the molecule of tRNA.

These are:

(a) Amino acid attachment site.

(b) Recognition site.

(c) Anticodon or codon recognition site.

(d) Ribosome recognition site

RIBOSOMAL RNA or rRNA

283

The RNA, which is found in ribosomes, is called ribosomal RNA. Ribosomes

are chemically ribonucleoprotein as they consist of RNA and proteins. It is

known as soluble RNA. Its quantity in a cell is much higher than that of

mRNA and tRNA. It constitutes about 80% of total RNA. On the basis of their

sedimentation coefficient or rate of sedimentation, rRNA molecules may be

classified into following categories:

28S-rRNA: It has molecular weight more than 10,00000. Sedimentation

coefficient is between 21S and 29S. It is found in 60S subunit of eukaryotic

ribosomes.

18s-rRNA: It molecular weight is less than a millions. Sedimentaion varies

between 12S to 18S. It is found in 40S subunit of ribosomes.

5S-rRNA: It has much lower molecular weight and is found in 30S unit of

ribosomes.

Structure of rRNA

Ribosomal RNA molecules are single stranded but in the solution of high

ionic concentration, irregular spiral coiling of rRNA is formed. As the ionic

concentration of the solution increases, the degree of irregular coiling of

rRNA also increases. In this coiling the intramolecular bases show base

pairing. The pairing is normal as A pairs with U and C pairs with G.

284

Check your progress-2

1. Note :- Write your answer in the space given

2. Compare your answer with the one given at the end of the

unit.

Write notes on

A. DNA replication

B. Protein Synthesis

--------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------

285

--------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------

--------------------------------------------------------------------------------------------

3.8 REPLICATION OF DNA

Semi-conservative replication of Chromosomes in eukaryotes:

Autoradiography experiment in Vicia faba, by J.H. Taylor and his co-workers

for the study of duplicating chromosomes in the root tip cells were first

published in 1957. They reported that DNA in all the organisms has the

inherent capacity of self-replication. The mechanism of DNA replication is so

precise that all the cells derived from a zygote contain exactly similar DNA

both in terms of quality and quantity. The replication takes place in

interphase after every cell division.

Theoretically, there may be following three possible modes of DNA

replication:

Dispersive Method

Conservative Method

Semiconservative Method

Semiconservative mode is the most accepted of all.

Semiconservative Method: - During replication, the two strands of the DNA

molecule uncoil with the help of some proteins and enzymes. The unpaired

bases in the single stranded regions of the two strands binds with their

complementary bases in the single stranded regions of the two stands bind

with their complementary bases present in the cytoplasm in the form of

286

nucleotides. These nucleotides become joined by phosphodiester linkages

generating complementary strands on the old ones. This provides for an

almost error free, high fidelity replication of DNA.

Fig. Semiconservative replication.

Detailed mechanism of semiconservative mode of DNA replication was given

by Kornberg. He proposed that following enzymes are important for

functional replication of DNA.

287

Nucleases

Unwinding Proteins

DNA Polymerases

DNA Ligases

RNA primer

Primases or RNA polymerases.

Out of the above six enzymes/proteins, the three; nucleases, RNA

polymerases and DNA polymerases are known as “Kornberg Enzyme”.

1. NUCLEASES

These are the enzymes which digest or breakdown nucleic acid molecules.

These attack on phosphodiester bonds of the nucleic acid backbone and

release nucleotides through hydrolysis. On the basis of their mode of

function, nucleases may be classified into following two:

(i) Exonucleases

(ii) Endonucleases.

Exonucleases function on phosphodiester bonds of the DNA at both the

terminus. The endonucleases, on the other hand attack the phosphodiester

bonds at intercalary regions of the DNA breaking it into as many parts as the

site of function.

2. UNWINDING PROTEINS.

Unwinding proteins are those proteins or enzymes, which uncoil the DNA

helix and separate the two DNA strands by breaking hydrogen bonds

between them. Due to this function these are known as unwinding proteins.

288

Due to separation, the two strands form a „Y‟ or fork like structure, which is

known as replication fork. Usually two types of unwinding proteins have been

recognized:

(i) DNA helicases : These enzymes or proteins uncoil the helix of DNA which

may now appear as ladder.

(ii) DNA gyrases: These enzymes breakdown the hydrogen bonds (A=T,

C=G) between two strands of a DNA molecule. E.g., DNA topoisomerase.

3. DNA Polymerases or Replicase Enzyme

DNA polymerases are the first enzymes suggested to be implicated in DNA

replication. It mainly function in the polymerazation of nucleotides on the

DNA template producing thereby the polynucleotide chain.

In eukaryotic cells, these three enzymes have been name as DNA

polymerase, β-DNA polymerase and -DNA polymerase. Due to their role in

DNA replication, DNA polymerases are also known as DNA replicases.

(1) DNA POLYMERASE-I: This enzyme was first discovered by Arthur

Kornberg in E.coli. After the name of the scienctist, DNA polymerase-I is also

known as „Kornberg enzyme‟ or „Kornberg polymerase‟. It is the most

extensively studied DNA polymerase studied in DNA replication machinery of

E.coli. Nowadays, it is believed that this enzyme is not responsible for DNA

replication and it mainly function in DNA repair.

Structurally, a molecule of DNA polymerase-I consists of a single polypeptide

chain having the molecular wt. 109,000. An atom of Zn is associated with

each molecule of this enzyme.

Following sites have been reported to be present on its surface:

289

(a) Template Site: it is occupied the DNA strand.

(b) Primer Site: The site at which the growth of DNA chain occurs.

(c) Nucleotide triphosphate site: The site where the incoming nucleotide

triphosphate is received.

(d) Primer terminus site: This site, which is used for removing any

mismatched nucleotide at the end of growing chain.

(e) Site for 5’ 3’ cleavage: The site which is used for removing any strand

coming in the path of growing primer. DNA polymerase-I function in following

activities within cells.

(i) 5’ 3’ polymerization activity: Attachment of nucleotides with each

other by the activity of DNA polymerase forming a polynucleotide chain is

called polymerization. The rate of polymerization in E.coli at 370 C has been

noted to be 1000 nucleotides per minute. It occurs in 5‟ 3‟ direction. It

forms small DNA segments through polymerization, which is used in repair

mechanism.

(ii) 3’ 5’ Exonuclease Activity: The activity of DNA polymerase-I is

performed to remove any nucleotide which mispair during elongation of

growing strand.

(iii) 5’ 3’ Exonuclease Activity: This activity is performed by this enzyme

to remove any DNA segment which comes as an obstruction in the way of

growing DNA Strand.

(iv) Removal of Thymine Dimmers: DNA polymerase-I function in the

removal of thymine dimers from the DNA strand. Such thymine dimmers are

produced to UV irradiation. After removal of diers, it also fills the gap, formed

dut to excision.

290

(2) DNA Polymerase-II : This enzyme has also been isolated from E.coli and

has the molecular wt. 90,000. Polymerization rate DNA polymerase-II is

much slower than polymerase-I. It is only 50 nucleotides per minute in E.coli,

it has 3‟ 5‟ exonuclease activity but lacks 5‟3‟ exonuclease activity unlike

polymerase-I.

(3) DNA- Polymerase-III : This is the main DNA polymerase involved in DNA

replication. This polymerase enzyme was originally discovered in a lethal

mutant of E.coli having mutation at dnaE locus. The enzyme has higher

affinity for nucleotides than polymerase-I and II have. The rate of

polymerization by polymerase-III is approximately 10-15 times higher than

the polymerase-I.

Structurally, DNA polymerase molecule consists of two polypeptide chains

each having the molecular wt. of 90,000. This dimeric enzyme does not

function unless it associates with two more chains of copolymerase-III each

having the molecular wt. of 77,000. The holoenzyme may be represented as

α2β2 where α2 polymerase-III and β2 copolymerase-III. ATP is also needed for

the growth of polynucleotide chain.

291

Fig.- Enzyme polymerase.

The enzyme DNA polymerase-III has 3‟5‟ exonuclease activity and 5‟3‟

polymerase activity.

4. DNA LIGASES

Through polymerase activity by DNA polymerase-III, polynucleotide chains is

formed in the form of small fragments which are known as Okazaki

fragments. In order to form a complete chain complementary to that of the

template, ligation of okazaki fragments is essential. The ligation reaction is

performed by RNA ligases.

5. RNA PRIMER

DNA replication really starts with the formation of a RNA fragment known as

RNA primer. It is formed at the point of origin. The primer is formed through

polymerization activity by RNA polymerase. Due to this reason, RNA

polymerase is also needed for functional replication of DNA.

6. PRIMASES

This is the group of enzymes, which are involved in RNA primer synthesis.

RNA polymerase is an example of primases.

Detailed molecular biological studies of the process of DNA replication now

have revealed that the process is much complex and requires a multi-

enzyme complex. About two dozens of enzymes are involved in this

complex. The complex is known as „replisome‟

STEPS OF DNA REPLICATION

292

Before studying the mechanism of DNA replication, we must be familiar with

following terms:

1. Replicon: A replicon is the unit of DNA in which individual act of

replication takes place. It has capacity of DNA replication independent of

other segments. Therefore, each replicon has its own origin and terminus, at

which DNA replication stops.

2. Origin: This is the sequence of a replicon which supports initiation of DNA

replication and also regulates the frequency of replication initiation. A general

feature of origin is that it is A=T rich. An origin in E.coli, oric has been

identified to 250 base pairs long.

3. Terminus: In most of prokaryotes, replicons has a specific site at the

extreme downstream of the strands. This site stops replication fork

movement and thereby terminates DNA replication.

In order to understand the exact mechanism of DNA replication. The process

must be studied in stepwise manner.

The overall process is completed in following steps:

(1) Before the start of DNA replication and formation of origin point, the

enzyme DNA helicase associates with the site of DNA. Its molecules unwind

the two strands of the DNA. Another enzyme, DNA gyrase or DNA

topoisomerase breaks the hydrogen bonds between the two strands and

separate them from each other forming a „Y‟ shaped replication fork.

(2) The two strands of a DNA molecule separated in the way explained in the

first step function as template. It should be noted here that template is the

single strand of DNA on which polymerization of nucleotides forming a new

strands takes place.

293

In eukaryotes, evidences for bi-directional DNA replication are

available. DNA replication starts at many points each of which start as a loop

and can be seen as expanding bubbles or eyes in electron micrograph.

The number of eyes or bubbles indicates the number of replicons.

Figure- DNA Replication

(2) Formation of RNA Primer: Before the actual replication of DNA starts at

origin, a short fragment of RNA is synthesized with the help of RNA

polymerase. This RNA fragment is called RNA primer. It is believed that it

provides safety to the new DNA strands, which is synthesized extending the

RNA primer itself.

294

(3) Synthesis of Complementary Strand of DNA: DNA replication or

synthesis of a new DNA strand complementary to the template is catalysed

by the enzyme, DNA polymerase-III. It starts at the end RNA primer in 5‟3‟

direction. The nucleotide sequence in the new strand is always

complementary to the sequence of nucleotides in the template.

Some features of DNA replication are as follows:

(i) DNA Replication is Bi-directional: Johan Cairsn on the basis of his

experiments on atoradigraphy concluded that DNA synthesis starts at a fixed

point on the chromosome and proceeds in one direction only. Subsequently,

it was realized that Cairns results could be interpreted in terms of bi-

directional replication also. On the basis of many other experiments it has

convincingly been demonstrated that DNA replication begin at a unique site

at origin and proceeds in both the direction on a strand. It takes place in the

form of pieces called „Okazaki fragment which are joined with each other with

the help of DNA ligases.

(ii) The two strands of the parent DNA at the point of replication fork or origin

replicate together with each other.

(iii) Replication of 3‟5‟ strand of DNA molecule is continuous and the new

strand grows in 5‟3‟direction. Replication in the second strand of the DNA

molecule is discontinuous. Replication of this strand starts somewhat later

than that of strand. Consequently, a given segment of 53‟ strand always

replicates i.e., the 3‟5‟ strand. Therefore, the 3‟5‟ strand of the parent

DNA molecule is known as the leading strand while the 5‟3‟ strand is

termed as the lagging strand.

295

(iv) Formation of RNA primer takes place in the beginning of each and every

okazaki fragments.

4. Termination of DNA Replication: The termination of DNA replication so

signaled by specific sequences, the ter-elements. E.coli, the ter-element of

R6 K plasmid has a 23 base pair sequence. This site functions as the binding

site of Tus, a 36 K dal protein necessary for termination. This stops the

replication fork movement and thereby stops DNA replication.

5. Removal of RNA Primer: After the whole DNA molecule is replicated on

both the strands, the RNA primer on all the segments are removed or

degraded with the help of DNA polymerase-I through its nuclease activity.

6. Synthesis of DNA Strand at the place of RNA Primer: in order to have

replication of a complete DNA molecule, replication of the segment at the

place of RNA primer is necessary. This process is performed with the help of

DNA polymerase-I instead of polymerase-III through polymerization of

deoxyribonucleotides. After DNA replication at the place of RNA primer, the

replication process is completed.

7. Proof Reading and Repair Mechanism: The complementary base

pairing during DNA replication is much accurate and precise, however, there

are chances of error in this process of base pairing. It is mainly due to

various physical and chemical forces involved in rate of error in E.coli are 5 x

10-8 to 5 x 10-10.

8. The proof reading of newly synthesized DNA strand is done by DNA

polymerase-III through correction of mismatched base pairing. It deletes the

wrong base and replaces it by a correct base. The process of proof reading

296

also takes place in 5‟3‟ direction. In E.coli, mutants defective in proof

reading show an increase in mutation frequency by over 1000 times.

3.9 TRANSCRIPTION

The production of RNA copies from a DNA template is known as

transcription. It is catalysed by a specific enzyme RNA polymerase or

transcriptase. During this process, only one strand of DNA duplex is known

as template strand or antisence strand. This results into the production of m-

RNA molecule having base sequence complementary to the template DNA

strand. It should be noted here that the sense. strand or coding strand of

DNA is now copied and has the same base sequence as the RNA produced

by the antisense strand.

The RNA polymerase is a complex enzyme and usually consists of a larger

protein part (apoenzyme), which is known as core enzyme and a cofactor,

which is known as sigma factor. The two combines to produce the complete

enzyme of holoenzyme. Unless and until the two parts of RNA polymerase

do not combine with each other, it is not functional. As far as the nature of

RNA polymerase in prokaryotes and eukaryotes is concerned, it shows much

diversity. While in prokaryotes like E.coli a single species of this enzyme is

found, at least three distinct RNA polymerases have been reported in nuclei

of most of eukaryotes. These have been named as : 1. RNA polymerase-I or

A, 2. RNA polymerase-II or B and 3. RNA Polymerase-III or C. They have

different functions as:

RNA polymerase-I or A: It is located in the nucleolus and responsible for the

synthesis of rRNA.

297

RNA polymerase-II or B: It is found in the nucleoplasm and is responsibe

for the synthesis of hnRNA which is a precursor of mRNA.

RNA polymerase-III or C: It is also found in the nucleoplasm. It is

responsible for the production of 5s rRNA and tRNA.

1. A Promoters for RNA Polymerase. Promoters for RNA polymerase I

have atleast two elements:

A GC-rich upstream (-180 to -107) control element.

A core region that overlaps the transcription start site (-45 to +20).

Protein coding structural genes in higher eukaryotes are transcribed in the

nucleus, but the primary RNA transcripts in the nucleus differ from mRNAs

used in the cytoplasm for translation. The RNA transcripts in the nucleus are

collectively described as heterogeneous nuclear RNA or pre-mRNA

molecules each of which is generally much larger than its corresponding

mRNA. The hnRNA molecules, which are destined to produce mRNA,

undergo RNA processing which includes the following events: (i) Modification

of 5‟ end by capping and modification of 3‟ end by a tail after enzymatic

cleavage; (ii) Splicing out of intron sequences from RNA transcripts of

interrupted genes. Cleavage and polyadenylation usually proceed RNA

splicing.

Promoter, enhancer and silencer sites for initiation of transcription in

eukaryotes

In eukaryotes there are three RNA polymerases: RNA polymerase I or

RNAPI for synthesis of pre-rRNA; RNA polymerase II or RNAPII for synthesis

of re-mRNA or hnRNA and several snRNAs, and RNA polymerase III or

298

RNAPIII for synthesis of 5S RNA, tRNA. Different promoter sequences have

been identified for different RNA polymerases.

MECHANISM OF TRANSCRIPTION: The overall process of transcription is

completed in following steps:

Formation of holoenzyme: The core enzyme of RNA polymerase cannot

start the polymerization process producing RNA. It first combines with the

sigma factor and produce the holoenzyme, It is assumed that the sigma

factor helps the enzyme in recognition of the initiation site on the DNA

template.

Attachment of holoenzyme on DNA duplex: The holoenzyme first binds at

the promoter site of DNA forming the closed promotor complex or „closed

binary complex‟. In this stage the DNA still remains in the form of double

helical.

Unwinding of DNA: It includes strand separation in the DNA duplex in a

stretch of the DNA bound with RNA polymerase; It extends just beyond the

start point so that the template becomes available for transcription initiation.

The open DNA strands form the „open binary complex‟

Synthesis of RNA: After the open binary complex is formed on DNA,

synthesis of RNA starts. Once the template or antisense strand of DNA

becomes available, the enzyme begins to incorporate RNA nucleotides

beginning at the start points. The polymerization of these nucleotides takes

place in 5‟ 3‟ direction. As the enzyme molecule move ahead in this

direction, phosphodiester linkage or bond is formed between two adjacent

nucleotides.

299

The process of elongation of RNA synthesis take place when the

holoenzyme leave the promoter region and move ahead in 5‟3‟ direction.

Together with the movement of the holoenzyme, the trancription bubble also

moves in the same direction. The transcription bubble represents the region

of the DNA duplex in which the two strands are separated from each other.

The length of the bubble ranges from 12 to 20 base pairs. The bubble

movement and sequential adding of correct nucleotides on RNA chain take

place simultaneously. The 5‟ end of the newly synthesized RNA

progressively separate from the DNA template DNA. In the back of the

bubble, the two DNA strands reassociate to form DNA duplex.

Termination of RNA formation: Specially in prokaryotes, termination of

transcription or RNA formation is brought about by certain termination signals

on DNA The termination may be of two types:

Rho Independent Terminations: This types of RNA synthesis termination is

due to specific sequences on DNA. A typical hairpin like structure is formed

on DNA template due to which the movement of RNA polymerase on the

template is obstructed. The hairpin structure is formed due to inverted repeat

sequences on DNA. The hairpin or stem-loop is followed by a run of adenine

residues in DNA and U residues in mRNA in the downstream.

Rho Dependent Termination: This type of termination is due to presence of

special factor, which is called Rho factor. It has a mol. wt. of 60,000 and is

not a part of RNA polymerase. After the synthesis of mRNA on template DNA

is completed, it attaches with the template. The site for its attachment is

characterized by 5‟-CAATCAA-3‟. The actual and precise mechanism of the

function of factor is not known.

300

In eukaryotes, the termination process is more completed. The termination

sites similar to prokaryotes are also operative in eukaryotes but these sites

are believed to be present away up to 1 kb from the site of the 3‟end of the

mRNA. AAUAAA sequence on mRNA and „snurp‟ are assumed to play

important role in termination of the process in eukaryotes.

Maturation of mRNA from hnRNA in eukaryotes: The mature mRNA

molecules very often have much lower molecular wt. and base sequence

length in comparison to the DNA segment from which it is transcribed. The

primary RNA transcript of a structural gene is called pre-mRNA. It is also

known as the heterogeneous RNA, high molecular wt. RNA. It is much bigger

in size than mRNA. The later is formed by splicing of hnRNA followed by

some other modifications. The heteronuclear mRNA undergoes following

modifications: -

Addition of Cap (m7G) and Tail (Poly A) for mRNA in Eukaryotes

Addition of methylated cap at the 5’ end

The initial RNA transcript, derived from genes coding for proteins, gets

modified so that its 5‟ end gains a methylated guanine and its 3‟ end is

polyadenylated. Capping at 5‟ end occurs rapidly after the start of

transcription and much before completion of transcription. Transcription

starts with a nucleoside triphosphate, and a 5‟ triphosphate group is retained

at this first position. The initial sequence at 5‟ end of hnRNA is therefore 5‟

pppApNpNp…3‟. To the 5‟ end is added a terminal G with the help of an

enzyme guanyl transferase as follow:

5‟Gppp+5‟pppApNpNp 5‟Gppp5‟ApNpNp+pp+p

301

The new G residue is in the reverse orientation with respect to all other

nucleotides and undergoes methylation at its 7th position. The cap with a

single methyl group at this terminal guanine residue is found in unicellular

eukaryotes and described as cap0, but in most eukaryotes, methyl group

may also be present on the penultimate base at 2‟ position of sugar moiety,

so that nucleotides, it is now described as cap1. Removal of cap leads to

loss of translation activity due to loss of the formation of mRNA-ribosome

complex. It suggests that the „cap‟ helps in recognition of ribosome. Only in

some eukaryotic nRNAs, caps may be absent and may not be required for

translation.

2. Polyadenylation and the generation of 3’ end in eukaryotes

The 3‟end of n RNA is generated in two steps (i) Nuclease activity cuts the

transcript at an appropriate location. (ii) Poly (A) is added to the newly

generated end by an enzyme, poly (A) polymerase (PAP), utilizing ATP as a

substrate. Ordinarily 30% of hnRNA and 70% of mRNA are polyadenylated.

In addition to AAUAAA, there are following consensus sequences, that are

involved in polyadenylation: (i) a G-U rich element is present downstream to

the site of cleavage, and is important for efficient processing for

polyadenylation: (ii) a G-A sequence immediately5‟ of the cleavage site;(iii)

consensus upstream element situated 5‟ of a poly A signal or AAUAAA.

Splicing of RNA parts coded by introns: Self splicing is a very common

phenomenon found in hnRNA. In this process generally those parts of the

RNA are removed or spliced out, which have been transcribed from intron

regions of the template DNA. These regions have short consensus

sequences which pair to formstem-loop like secondary structure. These are

302

helpful in self or autosplicing. Stem-loop like structures were observed for the

first time in the hnRNA of Tetrahymena thermophila.

Editing of RNA: Theoretically, the base sequence of a mRNA is just

complementary to the base sequence of the segment of the template DNA

from which it is transcribed. However, in many cases, the base sequence of

mRNA has been found to be changed after transcription at the level of RNA.

This process of change in the base sequence of mRNA is known as RNA

editing. It may be confined to a single base or may affect the entire mRNA.

3.12 RIBOSOME STRUCTURE, r-RNA AND BIOSYNTHESIS

Ribosomes are round, granular and membraneless cell organelle which are

chemically nucleoprotein and found enormously in all the prokaryotic and

eukaryotic cells. These were discovered first by Claude in 1943 and were

named as „microsomes‟. Robinson and Brown isolated ribosomes from root

cells of broad bean. Palade coined the term „ribosomes‟ and isolated it from

animal cells. After his name ribosomes are laso called „Palade granules.‟

Ribosomes may be defined as “The smallest known electron microscopic,

ribonucleoprotein particles attached the on RER or floating freely in the

cytoplasm and are the sites of protein biosynthesis”.

OCCURRENCE: Ribosomes are generally found in all known prokaryotic

and eukaryotic organisms except mature RBCs. In prokaryotes these are

found only in free form in the cytoplasm while in eukaryotes these are found

both in the cytoplasm and on the surface of RER. The former is called

cytoplasmic and later is called bound form of ribosomes. These may also be

found on the surface of nuclear membrane. Some organelles like

303

mitochondria and chloroplast contain ribosomes in the matrix. These are

called organellar ribosomes and reffered as „mitoribosomes‟ and

„plastidoribosomes‟. The ribosomes found on the surface of RER is bound

with the membrane with special proteins called ribo-phorines.

Number: The number of ribosomes in a cell depends on the content of RNA.

These are more in number in metabolically active cells like plasma cells,

livercells, nissl‟s granules of nerve cells, meristematic cells, cancer cells,

endocrine cells etc. In a cell of E.coli, the number of ribosomes vary from

10,000-20,000.

Structure: Ribosomes are globular structures having the diameter of 150-

250 Å. Each ribosome is made up of two subunits one is smaller and another

is larger in size. The later in dome shaped and is covered by cap like smaller

unit. In 70S type of ribosome the larger and smaller units are 50S and 30S

type. On the other hand, in 80S type, these are of 60S and 40S type,

respectively. The two subunits of ribosomes are freely distributed in the

cytoplasm. The two subunits unite to form a complete ribosome. Likewise,

the two subunits dissociate with each other when the concentration of Mg++

ion decreases in the cytoplasm.

During protein synthesis many ribosomes become attached with mRNA

forming a peculiar structure called polyribosome.

304

Location of antigenic sites of ribosomal proteins. Stoffler and Wittmann’s model

Ultrastructure of Ribosomes

The last point about the ultra structure of ribosomes has not been said till

date. The credit of giving the present knowledge of the ultrastructure of

ribosomes goes to Nauninga. According to him, the size of larger (50S)

subunit of 70S type of ribosome is 160 to 180 Å which is pentagonal in

305

shape. This unit has a groove of 40-60 Å size in which the smaller subunit is

attached during association. The smaller subunit has a platform, cleft, head,

base and also a binding site for nRNA. The smaller unit of 70S and 80S type

of ribosomes does not have a definite shape. Florendo in 1968reported a

pore like transparent area on the larger unit of 50S of 70S ribosomes.

In between two subunits of ribosomes, mRNA is found. t-RNA molecule is

found in the side of nRNA. The new-formed polypeptide chain being

synthesized on the ribosome mRNA complex has been seen passing through

the transparent pore on the larger unit. It also has a protuberance, a ridge

and stalk. Two binding sites, peptidyl and amino acyl sites are found on the

larger units.

The 50S and 30S subunits have been reported to have the molecular weight

of 1.8 X 106 Daltons and 0.9 X 106 Daltson, respectively. It must be noted

here that size and type of ribosomes and their subunit are determined on the

basis of their sedimentation coefficient.

Types of Ribosomes

On the basis of their sedimentation coefficient, ribosomes have been

classified into two main types:

70S ribosomes: These are found in prokaryotes and mitochondria and

plastids of eukaryotes. Each 70S ribosome is about 200-290 Å in size and

2.7 X 106 Daltons in its mol. Weight. It consists of two subunits of 50S and

30S size. Both of these units are made up of ribosomal RNA and ribosomal

proteins. The 50S subunits again consist of 23S and 5S rRNA and 30 types

of proteins. Similarly, the smaller unit is made up of 16S type of rRNA and 20

types of proteins.

306

80S ribosomes: These are the characteristic of eukaryotic cells and found.

In their cytoplasm. It consists of two subunits. The size of larger subunits is

60S and that of smaller subunit is 40S. It is also made up rRNA and proteins.

The 60S subunits consists of 28S rRNA, 5.8 S rRNA and 5S rRNA and about

50 types of proteins. The smaller subunits is similarly made up of 18S rRNA

and 30 different proteins.

Polyribosomes or Polysomes: When many ribosomes are attached to

same mRNA strand, it is called polyribosomes or polysome.

It is formed when a simple protein is required in high quantity. The number of

ribosomes in a polysome depends on the length of mRNA. The distance

between two adjacent ribosome is about 90 nucleotids.

Origin of ribosomes:

We have studied that ribosomes are solely made up of rRNA and proteins.

The former is formed inside the nucleus and the later is produced in the

cytoplasm. Therefore, these are partly nuclear and partly cytoplasmic is

nature. However, in prokaryotes, since there in no nucleus, ribosomes are

totally cytoplasmic in nature.

Functions of Ribosomes.

Ribomes are called factories of proteins or engineers of the cell because

these are the side of protein synthesis.

Sometimes rRNA of ribosomes has been found to function as enzymes

controlling the cellular functions. These are called ribozymes.

The process of translation of genetic language into the language of enzymes

or protein take place at ribosomes. It takes place with the help of rRNA,

which, is produced during transcription of nuclear DNA.

307

In general the ribosomes bound on RER synthesise enzymes for

extracellular use e.g., pancreatic cells, chief cells of gastric glands, liver cells

etc.

Ribosomes temporarily store proteins.

Ribosomes keep the mRNA molecules functionally alive.

RIBOSOMAL RNA or rRNA

The RNA, which is found in ribosomes, is called ribosomal RNA. Ribosomes

are chemically ribonucleoprotein as they consist of RNA and proteins. It is

known as soluble RNA. Its quantity in a cell is much higher than that of

mRNA and tRNA. It constitutes about 80% of total RNA.

On the basis of their sedimentation coefficient or rate of sedimentation, rRNA

molecules may be classified into following categories:

28S-rRNA: It has molecular weight more than 10,00000. Sedimentation

coefficient is between 21S and 29S. It is found in 60S subunit of eukaryotic

ribosomes.

18s-rRNA: It molecular weight is less than a millions. Sedimentaion varies

between 12S to 18S. It is found in 40S subunit of ribosomes.

5S-rRNA: It has much lower molecular weight and is found in 30S unit of

ribosomes.

Structure of rRNA

Ribosomal RNA molecules are single stranded but in the solution of high

ionic concentration, irregular spiral coiling of rRNA is formed. As the ionic

308

concentration of the solution increases, the degree of irregular coiling of

rRNA also increases. In this coiling the intramolecular bases show base

pairing. The pairing is normal as A pairs with U and C pairs with G.

Function of rRNA

The main function of rRNA may be summarized as below:

In many viruses specially in plant viruses, RNA function as genetic material

and carry genetic information from generation to generation. Different RNAs

function as structural component of a cells mRNA are the site of protein

synthesis where polymerization of amino acids takes place through peptide

bond formation between amino acid molecules during translation process. A

tRNA molecule has anticodon site and has the capacity of attachment with

the complementary condon on mRNA. tRNAs further carry activated amino

acids to the mRNA and catalyse peptide bond formation between two amino

acid molecules.Ribosomal RNA(rRNA) is the constituent unit of ribosomes.

TRANSLATION AND GENETIC CODE

The synthesis of protein from mRNA involves translation of the language of

nucleic acids into language of proteins. For initiation and elongation of a

polypeptide, the formation of aminoacyl transfer RNAs is a prerequisite,

Formation of Aminoacyl rRNA

Activation of amino acid

This reaction is brought about by the binding about by the binding of an

amino acid with ATP and is mediated by specific activating enzymes known

as amino acyl tRNA syntehtases or aaRs. As a result of this reaction

between amino acid and adenosine triphosphate, mediated by specific

enzyme, a complex (amino acyl-AMP- enzyme complex) is formed. Amino

309

acyl-RNA synthetases are specific with respect to amino acids. For different

amino acids, different enzymes would be required.

Aa1 + ATP (Enzl) (aa1-AMP) Enz1 + PP

Activation of amino acid

The transfer of amino acid to rRNA

The amino acyl-AMP-enzyme complex, formed during the step outlined

above, reacts with a particular tRNA and transfers the amino acid to the

tRNA. A particular amino acid would require a particular enzyme and a

particular species of tRNA. This would mean that for 20 amino acids, at least

20 different enzymes and also atleast 20 different t-RNA species would be

required.

(aa1-AMP) Enz1 + t-RNA1 aa1- t-RNA1 + AMP + Enz1

310

Transfer of amino acid to tRNA

Initiation of Polypeptide

The initiation of polypeptide chain is always brought about by the amino acid

methionine, which is regularly coded by the condon AUG,

In eukaryotes, formylation of initiating methionine is not brought about due to

the absence of tRNAfmet in plants and animals. Initiation in higher organisms

will therefore, take place without formylation.

Initiation in eukaryotes

Initiation of polypeptide chain in eukaryotes is similar to that is prokaryotes,

except the following minor differences. (i) In eukaryotes there are more

initiation factors. They are named by putting a prefix „e‟ to signify their

eukaryotic origin. These factors are eIF1, eIF2, eIF3, eIF4A, eIF4B, eIF4C,

eIF4D, eIF4F, eIF5 and eIF6. (ii) In eukaryotes, formylation of methionine

does not take place. (iii) In eukaryotes, smaller subunit associates with

initiator tRNAimet, without the help of mRNA, while in prokaryotes, generally

the 30S-mRNA complex is first formed which then associates with f-met-

tRNAfmet.

Kozak’s ribosome scanning hypothesis for translation in eukaryotes

In 1983, Marilyn Kozak proposed a hypothesis for initiation of translation by

eukaryotic ribosome. According to this hypothesis, 40S smaller subunit of a

eukaryotic ribosome with its associated met-tRNA moves down the mRNA

from 5‟ end, until it encounters the first AUG. At this point, the 60S subunits

join and the translation begins. The 80S ribosome, after reaching termination,

releases protein and dissociates in two subunits.

Elognation of Polypeptide

311

The following three steps are important in the elongation process.

Binding of AA-rRNA at site ‘A’of ribosome (classical vs hybrid state models for translation)

In earlier classical model, each ribosome had two cavities, in which tRNA

could be inserted. These were ‘P’ site and ‘A’ site. However, later a third

cavity was suggested. F-met-tRNAfmef comes on ‘E’ site, to make „A‟ site

available for the next amino acyl tRNA (AA-rRNA).

Various steps of protein synthesis: (A-B) Attachment of tRNA-fmet-mRNA

and smaller unit of ribosome, (C) Union of subunits of ribosomes, (D) Union

of second. Another site called „R‟ sites located on smaller subunit of

ribosome, was proposed „R‟ site plays a role in the improvement of accuracy

312

of translation. The aminoacyl rRNA first binds to R site involving codon-

anticodon pairing. Later aminoacyl rRNA is flipped to „A‟ site using energy

from GTP molecule. During this flipping, tRNA is held only by condon-

anticodon pairing. After formation of 70S initiation complex, the next amino

acyl tRNA enters „A‟ site. Elongation factors EF-Tu and EF-Ts participate.

The elongation factor EF-Tu first combines with GTP and changes to an

active binary complex, which binds with aa-tRNA, to form a ternary complex.

EF-Tu-Ts+GTP EF+Tu.GTP+EF-Ts

Binary Complex

EF-Tu.GTP+aa-tRNA EF-Tu..GTP.aa-tRNA

(Ternary Complex)

(Hybrid states Models), it has been shown that above ternary complex

actually binds in an A/P hybrid state, the anticodon binding to the A-site of

the 30S subunit and the CCA end binding to the P-site of the 50S subunit as

well as to the 30S subunit. The „P‟ site is already occupied by f-met. tRNAfmet

or by a peptidyl tRNA. Following the GTP hydrolysis, EF-Tu.GDP+P are

released from the ternary complex, permitting movement of CCA end of aa-

tRNA into A site of the large 50S subunit. EF-Ts now displaces GDP in the

EF-Tu.GDP binary complex and associates with EF-Tu, so that GTP can

again associate with EF-Tu to start another cycle for the binding of aa-tRNA.

Formation of peptide bond

This is a catalytic reaction during which a peptide bond is formed between

the free carboxyl group of the peptidyl tRNA at the „P‟ site and the free amino

group present with amino acyl tRNA, which is available at the A site. The 50S

313

rRNA have peptidyl transferase activity, so that the ribosome is described

as a ribozyme.

According to this displacement model, peotidyl chain remains in a constant

position relative to ribosome, while the tRNA moves during the peptide

reaction. After the formation of peptide bond, the tRNA at „P‟ site is

deacylated and the tRNA at „A‟ site now carries the polypeptide.

Translocation of peptidyl tRNA

From ‘A’ to ‘P’ site.

The peptidyl tRNA present at ‗A‘ site is now Translocated to ‗P‘ site. For

translocation of peptidyl tRNA from ‗A‘ site to P site, there are two models

available: (i) According to two sites model, deacylated tRNA is liberated from ‗P‘

site, and with the help of one GTP molecule and an elongation factor EF-G, the

peptidyl tRNA is translocated from ‗A‘ to ‘P‘ site. Thus according to this model,

tRNA is either entirely in the A site or entirely in the P Site

314

Different stages in translation

The requirement of EF-G and GTP for translocation was revealed by the use of

antibiotic. The elongation factor EF-G binds to ribosome and is released on

hydrolysis of GTP, which is a ribosomal function. EF-G and EF-Tu cannot bind to

ribosome simultaneously, so that the entry of a fresh aa-tRNA on ‗A‘ site and the

translocation of peptidyl tRNA from ‗A‘ to ‗P‘ site has to follow each other and

cannot occur simultaneously.

315

In eukaryotes, the elongation factor needed for translocation is called eEF-2, for the

formation of one peptide bond. One ATP molecule and two GTP molecules (one for

transfer of aa-tRNA to ‗A‘ site and the other for translocation of peptidyl tRNA

from ‗A‘ to ‗P‘ site) are required.

Termination of Polypeptide

Terminations in mRNA with stop condon

Termination of the polypeptide chain is brought about by the presence of any one of

the three combination condons, namely UAA, UAG and UGA. These termination

condons are recognized by one of the two release factors RF1 and RF2. The release

factors to act on ‗A‘ site, since suppressor rRNA capable of recognizing by entry at

‗A‘ site. A third release factor RF3 stimulate the action of RF1 and RF2 in a GTP-

dependent and condon independent manner GTP molecule is hydrolysed during

release of a polypeptide, when RF3 stimulates RF1 and RF2. For release reaction,

the polypeptidyl tRNA must be present on ‗P‘ site and the release factors help in

splitting of the carboxyl group between the polypeptide and the last tRNA carrying

this chain. Polypeptide is thus released and the ribosome dissociates into two

subunits with the help of ribosome release factor or RRF.

It has been shown that the translation apparatus in chloroplasts and mitochondria

differs from that in cytoplasm in eukaryotes in the following respects. (i) Ribosomes

in these organelles are smaller in size than these in cytoplasm. (ii) The tRNAs are

specific and differ, the number of tRNAs in mitochondria being 22 as against 55 in

cytoplasm. (iii) Initiation of translation takes place by formyl-methionyl tRNA both

in chloroplasts and mitochondria, although no formylation takes place in cytoplasm.

(iv) Translation in chloroplasts and mitochondria can be inhibited by

chloramphenicol.

316

Modification of Folding of Released Polypeptide

Modification of released polypeptide

After translation, the released polypeptide is modified in various ways.

Due to the action of certain other enzymes, exo-amino-peptidases, amino acids may

be removed from either the N-terminal end or the C-terminal end or both.

The polypeptide chain singly or in association with other chains also folds into a

tertiary structure. This problem of protein folding is sometimes described as ‗Second

Half of the Genetic Code‘.

Genetic code :

Several theories were proposed to explain the mechanism by which a particular

sequence of nitrogenous bases in DNA by transcribing complementary bases in

mRNA determines the position of specific amino acid in the protein molecule. The

theory which is widely accepted till now was proposed by F.H.C. Crick. The theory

holds the existence of a genetic code and its smallest unit which codes for one amino

317

acid is known as codon. A codon (code word) is the nucleotide or nucleotide

sequence in mRNA which codes for particular amino acid, whereas the genetic code

is the sequence of nitrogenous bases in mRNA molecule, which encloses

information for the synthesis of protein molecules.

Essential features of genetic Code.

1. Triplet : A codon of the modern genetic code comprises of three nitrogenous

bases of mRNA in a specific sequence.

2. Commaless : There is no punctuation (comma) between the adjacent codons i.e.,

each codon is immediately followed by the next codon with no intervening spaces

of letters for comma.

3. Non-overlapping : Under the overlapping triplet code the number of codons

could be reduced to twenty. But evidences have been gathered in support of the

existence of non-overlapping code.

4. Ambiguity : The genetic code inside the cell medium (in vivo) is said to be

nonambiguous, because a particular codon always codes for the same amino acid. No

doubt the same amino acid may be coded by more than one codon (degeneracy), but

one codon never codes for two different amino acids.

5. Universality : The same genetic code is said to be present in all kinds of living

organisms including viruses, bacteria, unicellular and multicellular organisms.

6. Collinearity : The codons in DNA and mRNA and the corresponding amino acid

residues in the polypeptide chain have a linear arrangement which has been

demonstrated by the studies of T4 mutants. These produce incomplete head

protein molecules. These mutants can be shown to map in linear sequence by

recombination technique. This suggests that the code is collinear.

318

The multiple system of coding is known as degenerate system or degenerate

code and provides protection to organisms against many harmful mutations. The

major degeneracy occurs at the third position(3‘ end of the triplet codon). When

first two bases are specified, the same amino acid may be coded for whether the

third base is U, C, A, or G. This base is described as ‘Wobbly base’.

Indian born biochemist, Dr. H.G.Khorana, devised an ingenious technique for

artificially synthesizing mRNA with repeated sequences of known nucleotides.

For this valuable contribution he was awarded Noble Prize in 1968.

319

By using synthetic DNA, Khorana and his coworkers prepared chains of

polyribonucleotides with known repeating sequences of two or three nucleotides

as follows;

(a) Poly CUC UCU CUC UCU…………….

(b) Poly CUA CUA CUA CUA……………

In first case CUC and UCU are two codons arranged alternately in the

polynucleotide chain. This dictates the formation of polypeptide chain having two

amino acids (leucine and serine) arranged alternately. The second case is an

example of homopolymer chain. The polynucleotide chain is formed of repeated

linkage of codon CUA. This dictates the formation of a polypeptide chain

consisting of only one amino acid leucine.

KEY TERMS

Genetic code codon Triplet codon

Termination codon Initiation codon Ambiguity of genetic code

Degeneracy Wobble hypothesis

The multiple system of coding is known as degenerate system or degenerate

code and provides protection to organisms against many harmful mutations. The

major degeneracy occurs at the third position(3‘ end of the triplet codon). When

first two bases are specified, the same amino acid may be coded for whether the

third base is U, C, A, or G. This base is described as ‘Wobbly base’.

Check your progress-2

1. Note :- Write your answer in the space given

2. Compare your answer with the one given at the end of the unit.

Write notes on

1. DNA replication

320

2. Protein Synthesis

321

1.12 LET US SUM UP:-

Two types of nucleic acids are found in the cells of all living organisms. These are:

1. Deoxyribonucleic acid - DNA

2. Ribonucleic acid - RNA

The Chemical analysis has indicated that DNA is composed of three different types

of compounds:

7. Sugar Molecule represented by a pentose sugar, the deoxyribose or 2‘-

deoxyribose.

8. Phosphoric Acid.

9. Nitrogenous Bases: These are nitrogen containing organic ring compounds.

These are of the following four types:

I. Adenine represented by – A

II. Thymine represented by – T

III. Cytosine represented by – C

IV. Guanine represented by – G

These four nitrogenous bases are separated into two categories:

Purines: These are two-ringed nitrogen compounds. Adenine and guanine are the

two purines found in DNA. Their structural formulae are represented in fig.2.

322

Pyrimidines: These are formed of one ring only and include cytosine and thymine

Nucleotides ( The Monomers of DNA)

A nucleotide is formed of one molecule of deoxyribose, one molecule of phosphoric

acid and one of the four nitrogenous bases. Since there are four nitrogenous bases,

there are four type of nucleotides namely:

9. Deoxyadenylic acid -Adenine + Deoxiribose + Phosphoric acid

10. Deoxyguanylic acid -Guanine + Deoxiribose + Phosphoric acid

11. Deoxycytidylic acid -Cytosine + Deoxiribose + Phosphoric acid

12. Deoxythymidylic acid -Thymine + Deoxiribose + Phosphoric acid

DNA Structure & Forms.

The Watson & Cricks model of DNA, actually describes B form of DNA.

Two Polynucleolide chains run antiparallely.

Purines and Pyremedines are on inside while phosphate and deoxyribose units

are on outside.

The diameter of Helix is 20 A0, Adjacent basee are separted by 3.4 A

0 along

the helix and are related by a rotation of 360o.

The helical structure repeats after ten residues at interval of 34 A0.

G = C & A = T are hydrogen bonds.

G always pairs with C & A with T

Replication of DNA: -Basic Rules of Replication

Replication is a semi conservative process.

Replication has direction. It could be unidirectional or bi-directional fork.

Unidirectional – Mitochondrial DNA, ø

Bidirectional - In E.coli & Eukaryotic Chromosome.

Replication starts at a unique point on bacterial and viral chromosome.

323

Replication of both strands proceed by the addition of nucleotide monomers in

the 5‘ 3‘directional

Replication starts in short discontinuous pulses.

Replication at the level of short fragments in initiated by the production of a

short segment of RNA to serve as a primer for DNA polymerase.

Replication of viral DNA is circular but progeny has linear DNA,

Replication time is 30 Minutes in E.Coli.

Enzymes of Replication

RNA Polymerase or Primerases

DNA Polymerase

Nucleases (Endonucleases & Exonucleases)

DNA Ligases

Restriction Enzymes

Swivelases

Unwinding enzymes and proteins

Steps: -

Binding of Unwinding proteins

Initiation of chain

Elongation

Termination

Binding or joining by ligases.

Transcription: -

Transcription is the synthesis of mRNA and stable RNA molecules from a

DNA Template by RNA Polymerase, using ribonucleotide triphosphates as

precursor.

324

Bacterial cells contain only on RNA Polymerase where as eukaryotes have

at least three viz. RNA Poly. I, II & III.

The three stages of transcription are

Initiation

Elongation

Termination

Promoters consist of conserved seq. necessary for the initiation of

transcription.

There are many different types of promoters.

The promoter sites for RNA polymerase I and II are located before the start

site for transcription.

RNA Polymerase III recognizes sites within the gene itself.

In eukaryotes the promoters sites for RNA Polymerases I and II are located at

the 5‘ end of the gene but the polymerase III promoter lies with in the gene.

RNA Polymerase II promoters have TATA box with consensus seq. of

TATAAA.

Main features of eukaryotic Transcription

The template for transcription is a completes of DNA and Protein

That has beaded appearance.

Eukaryotic RNA synthesis starts at precise promoter seq. As a

Result genes that is very actively transcribed and show ―fern leaf‖ or

―Christmas Tree‖ configuration in tRNA and lampbrush chromosome.

At any one time, only a very small fraction of the total chromatin is

transcribed.The nascent RNA gets associated with protein as it is

being transcribed, producing ribonucleus protein particles (RNP). In

eukaryotes the nuclear envelope introduces as barrier between

325

transcription and protein synthesis

Eukaryotic mRNA

Eukaryotic mRNA is metabolically stable as compared to mRNA of

pro. As they have comparatively longer half-life.

It is monocistronic.

The 5‘ end of blocked by 7-methyl –G.]

The 3‘ end of euk. mRNA ends with A poly A segment./ Eukaryotic

genes.

Frequently contain insertions of non-coding DNA. Heterogeneous

Nuclear RNAs are mRNA precursors containing intervening

sequences. Eukaryotic mRNA are associated with proteins

Translation: -

Main steps of translation are: -

1. Activation of amino acids

2. Transfer of amino acid to t-RNA.

3. Initiation of polypeptide chain

4. Elongation of polypeptide chain

5. Termination of polypeptide chain

I. Activation of amino acids

It includes screening of amino acids.

There activation at carboxyl gr.

Enzyme is amino acyl tRNA synthetase

II. Transfer of a.a to t-RNA

t-RNA are specific and named after amino acids

Ester bonds are formed between amino acids & t-RNA

III Initiation

326

In 1975 Anderson reported IF- MP, IF –M1, IF-M2A, IF- -

-M3.

Formylation of methionine does not occur in rule.

Reaction is catalyzed by transformylase enzymes

Initiation complex is formed by mRNA + 40S ribonucleus S.U + tRNA +

GTP + 3 Initiation factors.

Here, nmet-tRNA binds first to 40s sub unit. met RNA.

The P and A sites are located on 70s. subunit. Met RNA binds to the P site.

AU other tRNA as first build to A site, then shift to P site.

60s& 40s sub units join to form 80s subunit elongation of polypeptide

chain.

EF1& EF2 are required for elongation of polypeptide chair.

In the presence of elongation factor, amino acyl tRNA bends to A site (by

using GTP) on ribosome. Thus ternary complex is formed.

Peptide bond formation: -

First amino acid is now united by peptide bond formation with second

amino acids.

Peptide bond formation does not require external energy source.

This reaction is catalyzed by peptide transferase complex located in large

subunit

Alpha Amino group of one amino acid is bonded to the alpha- carboxyl of

other with elimination of H2O.

Translocation : -

The movement of ribosome relative to mRNA is called translocation.

It occurs in 53‘ direction.

327

Termination.

RNA polymerase recognizes termination signal UAA,UAG & UGA.

The termination condons provides signals to the ribosomes for attachment

of release factors. RF-1, RF-2, RF-3.

1.13 CHECK YOUR PROGRESS KEY

Check your progress Key-1

1. Purines are adenine , guanine,

Pyrimidines are Thymine , Uracil , Cytosine.

2. A+G/C+T = 1

3. Watson & Cricks model.

Check your progress Key-2

1. Rules of replication , enzymes of replication.

2. Transcription & Translation steps.

1.14 ASSIGNMENT / ACTIVITY

Q1. Explain process of DNA replication in eukaryotes.

Q2. Prepare a model of DNA.

Q3. Write short notes on

328

a. Transcription

b. DNA hydrolysis.

1.15 REFERENCES

1. E.D.P De Roberties & E.M.F. De Robertis Jr. - Cell and Molecular

Biology.

2. David Friefielder - Molecular Biology.

3. P.K. Gupta - Cell and Molecular Biology.

4. Stanier - Microbiology

5. Benjamin Lewin - Genes.

6. C.B.Powar - Cell Biology

.

329