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INTERMEDIARY METABOLISM

Introduction to Metabolism and Bioenergetics

Najma Z. Baquer Emeritus Professor

School of Life Sciences Jawaharlal Nehru University

New Delhi – 110 067

(02 April 2007)

CONTENTS

General features of metabolismCatabolic (degradative) and anabolic (biosynthetic) pathwaysExperimental approaches to study metabolismLaws of thermodynamicsFree energy changeStandard free energy change

Key words Heterotrophs, Autotrophs, Enzymes, Metabolism, Catabolic pathways, Anabolic pathways, Macromolecules, adenosine triphosphate (ATP), nicotinamide adenine dinucleotide phosphate (NADPH), Mutants, Isotopes, Endergonic, Exergonic, Intermediate, Oxidized, Reduced.

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General features of metabolism Myriad, of enzyme catalyzed reactions take place in living cells. Although we collectively refer to these reactions as “metabolism”, we must not think of cell metabolism in terms of a membrane-surrounded bag of randomly acting enzymes. Metabolism is a highly coordinated and purposeful cell activity, in which many multienzyme systems cooperate. Metabolism has four specific functions: 1. To obtain chemical energy from the degradation of energy rich nutrients from the

environment or from captured solar energy.

2. To convert nutrient molecules into the building block precursors of cell macromolecules. 3. To assemble these building blocks into proteins, nucleic acids, lipids, polysaccharides and

other cell components. 4. To form and degrade biomolecules required in specialized functions of cells.

Although metabolism involves hundreds of different enzyme catalyzed reactions, the central metabolic pathways, are few in number and they are identical in most forms of life. Living organisms can be divided into two large groups according to the chemical form of carbon they require from the environment.

(1) Autotrophic (self feeding) cells can use carbon dioxide from the atmosphere as the sole source of carbon and construct all their carbon containing biomolecules from it, e.g. photosynthetic bacteria and green leafy cells of plants. Some can also use nitrogen like cyanobacteria to generate nitrogenous components.

(2) Heterotrophic cells (feeding on others) cannot use atmospheric CO2 and obtain

carbon from their environment in the form of relatively complex organic molecules such as glucose. In higher animals and microorganisms, cells are mostly heterotrophic.

Autotrophic and heterotrophic organisms can be divided into subclasses. There are two classes of heterotrophic organisms - Aerobic and Anaerobic:

(1) Aerobes live in air and use molecular oxygen to oxidize their nutrient molecules. (2) Anaerobes live in the absence of oxygen. Many cells such as yeast, can live either

aerobically or anaerobically, such organisms are called facultative. Anaerobes that cannot use oxygen at all and indeed may be poisoned by it e.g. microorganisms, present in deep soils or the ocean floor, are called strict anaerobes.

“Enzymes” are the simplest units of metabolic activity, each catalyzing a specific chemical reaction. `Metabolism’, however consists of multienzymes sequences each promoting the sequential catalytic steps involved in a given metabolic pathway. Such enzyme systems may have anywhere from 2 to 20 enzymes, acting in a consecutive, linked fashion, so that the product

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of the first enzyme becomes the substrate of the second and so on. The “successive transformation products” in such a pathway [B, C, D, etc.] are known as metabolic intermediates or “metabolites”. Each of the consecutive steps in these pathways brings about a small specific chemical change, usually the removal, transfer or addition of a specific atom, a molecule or functional group. Through such orderly, step by step changes, the incoming biomolecule is transformed into its metabolic end product. Most metabolic pathways are “linear”, but some are “circular” or “cyclic”, usually metabolic pathways have branches leading in or out. The term “Intermediary metabolism” is often used to denote the specific sequences of intermediates involved in the pathways of cell metabolism.

Regulated step

E1 E15

E2 E14 E2

C L

E3

E13

D K

E4

E4 E12

E J

E5 E11 E5

Regulated step

A

B

E3

P

M

A

B

C

D

E

P

Catabolic (degradative) and anabolic (biosynthetic) pathways “Intermediary metabolism” has two phases : catabolism and anabolism. Catabolism is the degradative phase of metabolism in which organic nutrient molecules, e.g. carbohydrates, lipids and proteins coming either from the “environment” or the “cells own” nutrient stores, are degraded by stepwise reactions into smaller, simpler end products, e.g. lactic acid, CO2 and ammonia. Catabolism is accompainied by release of free energy inherent in the complex structure of large organic molecules. At certain steps in a catabolic pathway, much of the free energy is conserved, by means of coupled enzymatic reactions in the form of energy carrying molecule, adenosine triphosphate (ATP). Some may be conserved as energy rich hydrogen atoms carried by the coenzyme nicotinamide adenine dinucleotide phosphate [NADPH] in the reduced form.

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Release release of energy

Chemical Energy ATP NADPH

Catabolism (Degradative)

Anabolism (Biosynthesis)

Energy yielding nutrients Carbohydrates Fats

Cell Macromolecules Proteins Polysaccharides Lipids Nucleic Acids

Energy poor

End products CO2H2O NH3

Precursor molecules Amino acids Sugars Fatty Acids Nitrogenous bases

In anabolism also called biosynthesis, the building up or synthetic phase of metabolism, small precursor or building block molecules are built up into large macromolecular components of cells, such as proteins and nucleic acids. Since biosynthesis results in increased size and complexity of structure, it requires input of free energy which is furnished by the breakdown of ATP to ADP and phosphate. Biosynthesis of some cell components also requires high-energy hydrogen atoms, which are donated by NADPH. Catabolism and anabolism take place simultaneously in cells, and their rates are regulated “independently”. Catabolic pathways converge to a few end products The enzymatic degradation of each of the major energy-yielding nutrients of cells (carbohydrates, lipids and proteins) proceeds in a stepwise manner through a number of consecutive enzymatic reactions. There are three major stages in aerobic catabolism. In Stage I, cell macromolecules are degraded to their major building blocks. Thus polysaccharides are degraded to hexoses or pentoses; lipids are degraded to fatty acids, glycerol and other components, and proteins are hydrolyzed to their 20 component amino acids. In Stage II of catabolism, the various products formed in stage I are collected and converted into smaller number of yet simpler molecules. Thus the hexoses, pentoses and glycerol from stage I are degraded to a single 3-C intermediate Pyruvate, which is then converted into a single 2-C unit, the acetyl group of Acetyl-CoA. Similarly the fatty acids and the carbon skeletons of most

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of the amino acids are also broken down to form acetyl groups in the form of acetyl-CoA. Acetyl-CoA is thus the common end product of stage II of catabolism.

Stage 1 Pentoses

Stage II

Stage III

NH3

Fig. 1: The three stage

In Stage III the acetyl group of acpathway by which most energy yi(or other nitrogenous products) w It is important to note that the pastage III. The final pathway otributaries and streams.

Biosynthetic (anabolic) pathwayAnabolism, or biosynthesis, alsomolecules. For e.g. protein synprecursors. In the next stage the

Polysaccharides

Hexoses

Pyruvate

Citric acid cycle

COH2O

s of catabolism of major energ

etyl-CoA is fed into the citric aelding nutrients are ultimately ohich are the other end products o

thways of catabolism converge f catabolism thus resembles a

s diverge to yield many produ takes place in three stages, bethesis begins with the format α-ketoacids are aminated by a

Lipids

Proteins Amino acids Glucose Glycerol, fatty acids

Acetyl CoA

2

y-yielding nutrients

cid cycle - the “final” common xidized to CO2, water and NH3 f catabolism.

towards the citric acid cycle in widening river, fed by many

cts ginning with smaller precursor ion of α-keto acids and other mino group donors to form α-

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aminoacids. In the final stage of anabolism the amino acids are assembled into polypeptide chain to form many different proteins. Similarly acetyl groups are built up into fatty acids and they in turn are assembled to form various lipids. Just as catabolism is a converging process, anabolism is a diverging one, since it begins with a few simple precursor molecules from which a large variety of different macromolecules are made. Each major stage in the anabolism or catabolism of a given biomolecule is catalyzed by a multienzyme system. The sequential chemical changes taking place in the central routes of metabolism are virtually identical in all forms of life e.g. the catabolism of D-glucose to give pyruvic acid is accomplished through the same chemical intermediates and through the same number of reactions in most living organisms.

ATP carries energy from catabolic to anabolic reactions Complex nutrient molecules such as glucose contain potential energy because of their high degree of structural order. When it is degraded to CO2 and H2O much free energy becomes available. Free energy is that form of energy capable of doing work under constant temperature and pressure, this energy must therefore be captured and conserved or it will appear as heat. Much of the free energy released from glucose and other cellular fuels during their catabolism is conserved by the coupled synthesis of adenosine triphosphate (ATP) from adenosine diphosphate (ADP) and inorganic P.

CO2

O2

Fuels ADP +Pi

Catabolism

H2O 1. Biosynthesis 2. Contraction and Motility 3. Active Transport 4. Transfer of genetic

information

ATP

Energy dependent activities of cells depend upon transmission of energy by ATP. The energy conserved in the form of ATP can do four different kinds of work.

1. Biosynthesis, the terminal P group transferred to precursor molecules are “energized”. 2. Cell motility or contraction. 3. Active transport of nutrients and ions across membranes.

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4. Genetic information transfer during the biosynthesis of DNA, RNA and proteins.

Thus we have an energy cycle in cells, in which ATP serves as the energy carrying link between energy-yielding and energy requiring cellular processes.

NADPH carries energy in the form of reducing power A second way of carrying chemical energy from reactions of catabolism to the energy requiring reactions of biosynthesis is in the form of hydrogen atoms or electrons. When glucose is formed from CO2 during photosynthesis, or when fatty acids are made from acetate in the liver of an animal, reducing power in the form of hydrogen atoms is required for the reduction of double bond to single bonds, hydrogen atoms must have considerable free energy. Such high energy hydrogen atoms are obtained from cell fuels by dehydrogenases which catalyze removal of hydrogen atoms from fuel molecules and their transfer to specific coenzymes, particularly to the oxidized form of nicotinamide adenine dinucleotide phosphate (NADP+). The reduced or hydrogen carrying form of this coenzyme, designated NADPH is a carrier of energy-rich electrons from catabolic reactions to electron requiring biosynthetic reactions, just as ATP is a carrier of energy-rich phosphate groups.

Reduced fuel Catabolism Oxidized product NADP NADPH Carrier of energy electrons Reduced Reductive Oxidized Biosynthetic Biosynthetic Precursor Products reactions

Cell metabolism is an economical tightly regulated process Cell metabolism operates at maximum economy. The overall rate of energy yielding catabolism is controlled by the needs of the cell for energy in the form of ATP and NADPH. Thus cells conserve just enough nutrients to meet the energy utilization at any given time. Similarly the rate of biosynthesis of building block molecules and of cell macromolecules is also adjusted to immediate needs. Many animals and plants can store energy-supplying and carbon supplying nutrients, such as fat and carbohydrates, but they generally cannot store protein, nucleic acids, or simple building block molecules, which are made only when needed and in amounts required. Catabolic

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pathways are very sensitive and responsive to changes in energy needs, the regulatory mechanisms of central metabolic pathways, particularly those providing energy as ATP, are capable of responding to metabolic needs quickly and with great sensitivity.

Experimental approaches to study metabolism Three major approaches are used singly or in combination, to work out the chemical details of a metabolic pathway. In vitro studies The first and the most direct is to study the pathway in vitro (in glass i.e. in the test tube) in a cell free extract of a tissue capable of catalyzing the overall metabolic process e.g. conversion of glucose into ethanol and CO2 by yeast, was discovered by Buchner, 1898. Subsequently it was found that the breakdown of glucose in such extracts required the addition of inorganic phosphate, which disappeared from the extract as glucose was consumed. It was then found that phosphorylated intermediates (derivatives) of a hexose accumulated in the medium. Once the intermediates were identified, an enzyme was found in the yeast extract that acted upon it to form another product. Addition of enzyme inhibitors to yeast extract caused other intermediates to accumulate. By a combination of such approaches the 11 metabolites that are the intermediates in the conversion of glucose into ethanol in yeast were ultimately identified and isolated. Each of the 11 enzymes involved in this sequence have been isolated and identified. Many other metabolic pathways have been worked out by this direct approach in which the successive enzymes and intermediates degrading and forming them have been identified one by one. When the entire sequence is known it can be reconstituted in the test tube from purified components. Mutants of organisms allow identification of intermediate steps Another important approach to elucidation of a metabolic pathway is the study of genetic mutations of organisms, in which a given enzyme fails to be synthesized in active form. Such a defect, if not lethal, may result in the accumulation and excretion of the substrate of the defective enzyme. From genetic defects in specific enzymes occurring in people it has been possible for e.g., to deduce the nature of certain steps in the metabolism of amino acids. Such human genetic disorders are rare and do not lend themselves to systematic investigation. However, genetic defects in microorganisms can be produced at will by subjecting them to mutagenic agents e.g. irradiation with X-rays or treatment with certain chemicals, which can alter the structure of specific genes in their DNA. Such mutant microorganisms in which one enzyme or another is defective are powerful tools for study of metabolism. Isotopic tracers provide a powerful method of studying metabolism Another powerful method for establishing the general out line of a metabolic pathway is to use an isotopic form of an element to label a given metabolite. For example, the radioactive isotope 14C (the normal or average atomic weight of carbon is 12.01) is frequently used to label a specific carbon atom in an organic molecule. Such a 14C-labelled molecule is chemically indistinguishable from a normal labeled molecule, but can easily be detected and measured through its radioactivity. e.g. acetic acid can be synthesized in the laboratory in such a way that its carboxyl carbon atom is enriched in 14C which otherwise occurs in only extremely small and constant amounts in the carbon compound found in biosphere and geosphere.

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When such a sample of radioactively labeled acetate is fed to an animal, its metabolic fate can be readily traced. For example, the respiratory CO2 exhaled by the animal will be found to contain 14C indicating that some of the acetate is metabolized in such a way that its carboxyl carbon atom is converted into CO2. Moreover, if palmitic acid is subsequently isolated, from the liver lipids of the animals it will be also found to contain 14C indicating that the carboxyl carbon atom of acetate is a biosynthetic precursor of palmitic acid. Furthermore when the labeled palmitic acid molecule is chemically degraded it will be found to have excess 14C in only the alternate carbon atom beginning with the carboxyl carbon. H O H C-C* Acetate labeled in carboxylic atom H O-

O O Fed to rats and palmitic acid isolated from the liver

*C

CH2

*CH2

CH2

*CH2

CH2

*CH2

CH2

*CH2

CH2

*CH2

CH2

*CH2

CH2

*CH2

CH3

Figure 1: Use of an isotope of carbon to trace the metabolic fate of the carboxyl-C atom of acetate

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Much appears as exhaled 14CO2, significant amount is found in the palmitic acid of liver lipids isotope found in alternating C atom telling us that palmitic acid is made by 8 molecules of acetate joined in a head to tail fashion.

However if acetate containing 14C only in the methyl group is fed, again palmitic acid will be labeled, but in alternate carbon atoms starting from the α, or 2 carbon atoms. These observations led to the conclusion that all the carbons of palmitic acid ultimately derive from acetate molecules in a pathway that result in a head to tail linkage of the carbon skeleton of acetate molecules. Rate of metabolic processes The isotope tracer method can also be used to determine the rate of metabolic processes in intact organisms. One of the most significant advances made with this powerful method is the discovery that the macromolecular components of cells and tissues undergo constant metabolic turnover i.e. they exist in a dynamic steady state in the cell, in which constant biosynthesis is exactly counter balanced by an equal rate of degradation. For e.g. the isotopic measurements have shown that the protein of rat liver have a half life of about 5 to 6 days. On the other hand, the protein of skeletal-muscle or the brain turnover much more slowly.

Metabolic pathways are compartmented in cells 1. Prokaryotic and eukaryotic cells

Prokaryotic cells contain no compartments separated by internal membranes, yet there is some degree of segregation of certain enzyme systems in bacteria, e.g. the enzymes participating in the biosynthesis of proteins are located in the ribosomes and some of the enzymes participating in the biosynthesis of phospholipids are located in the bacterial cell membrane.

2. Animal or plant tissues

These are first gently homogenized in a isotonic sucrose medium, a process that ruptures the plasma membrane but leaves most of the internal organelles intact. Sucrose is used because it does not pass through membranes readily and thus does not cause internal organs such as chloroplast and mitochondria to swell.

The subcellular organelles, e.g. nuclei and mitochondria, which differ in size and specific gravity and thus sediment at different rates in a centrifugal field can then be isolated from the “homogenate” by differential centrifugation. The nucleic, mitochondria and other fractions obtained in this way can be tested for their ability to catalyze a given metabolic sequence. From this approach it can be found that different metabolic pathways take place in different intracellular locations in eukaryotic cells.

Cytosol glycolysis conversion of glucose lactate

Mitochondria TCA, electron transport

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Principle of thermodynamics Bioenergetics or Biochemical thermodynamics describes the transfer and utilization of energy in biological systems. It concerns only the initial and final energy states of reaction components, not the mechanism or how much time is needed for the chemical change to take place. In short, bioenergetics predicts if a process is possible. “Biological systems follow the general laws of thermodynamics.” Law of thermodynamics First law of thermodynamicsThe total energy of a system, including its surroundings, remains constant. This is also the law of conservation of energy. It implies that within the total system, energy is neither lost nor gained during any changes. Second law of thermodynamicsThe total entropy of a system must increase if a process is to occur spontaneously. SignificanceThus, Bioenergetics makes use of a few basic ideas from the field of thermodynamics, particularly the concept of free energy. Changes in free energy (∆G) provide a measure of the energetics feasibility of a chemical reaction and can allow prediction of whether a reaction will take place. The free energy concept is essential for understanding the unique role adenosine triphosphate (ATP) plays in transferring energy from energy yielding catabolic processes to energy – requiring reaction. ∆G: changes in free energy

• Energy available to do works. • Approaches zero as reaction proceeds to equilibrium. • Predicts whether a reaction is favourable.

∆H: changes in enthalpy • Heat released or absorbed during a reaction. • Does not predict whether a reaction is favourable.

∆S: Changes in Entropy • Measure of randomness. • Does not predict whether the reaction is favourable.

Relation between changes in free energy (G), Enthalpy (S) and Entropy (S).

∆G = ∆H – T∆S --------------------------------------------------- (I) Where T is the absolute temperature in degree Kelvin (oK): ok = bC + 273 Under the conditions of biochemical reactions, because ∆H is approximately equal, to ∆E, the total changes in internal energy of the reaction, the above relationship may be expressed in the following way: ∆G = ∆E – T∆S --------------------------------------------------- (II)

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A. Free energy change- Three varieties Go and ∆Go, ∆Go/

∆G- Is more general because it predicts the change in free energy and thus, the direction of a reaction, at any specified concentration of products.

∆Go- Is the change in standard free energy, when reactants and products are at 1mol/lt.

∆Go/- The standard free energy change at pH 7 i.e. in the standard state when the concentration of protons in assumed to be 10-7 mol/lt.

a. Sign of ∆G predicts the direction of a reaction: The change in free energy, ∆Go, can be

used to predict the direction of a reaction at constant temperature and pressure. Consider the reaction:

A B

A. Negative ∆G: if ∆G is negative, there is a net loss of energy, and the reaction goes spontaneously and the reaction is said to be exergoic.

B. Positive ∆G: if ∆G is positive, there is a net gain of energy. And the reaction does not go spontaneously and the reaction is said to be endergonic. The energy must be added to the system to make the reaction to go.

C. ∆G is zero: If ∆G = 0, the reactants are in equilibrium.

b. ∆G of the forward and backward reactions: The free energy of the forward reaction (A B) is equal in magnitude but opposite in sign to that of the back reaction (B A). For example, if ∆G of the reaction is –5000 cal/mol, then that of the back reaction is + 5000 cal/mol.

c. ∆G depends on the concentration of reactants and products: ∆G of the reaction A

B depends on the concentration of the reactant and product. At constant temperature and pressure, the following reaction can be derived:

[B] ∆G = ∆Go + RT In

[A] Where, ∆G is the standard free energy change.

R is the gas constant (1.987 cal/mol -degree) T is the abosulte Temperature (oK) [A] and [B] are the actual concentration of reactant and product. In represent the natural logarithm

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Fig.2: Change in free energy (∆G) during a reaction A. The product has a lower free energy (G) than reactants B. The product has a high free energy than the reactant

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d. Sign of ∆G can be different from that of ∆Go: A reaction with a positive ∆Go can proceed in the forward direction (have a negative overall ∆G) if the ratio [B]/[A] is sufficiently small (that is ratio of reactants to products is large). For example, consider the reaction:

(B) (A) (A) (A) (A) (A) (A) (A) (A) (A) ∆G = -0.96 Kcal/mol (B) (A) (A) (A) (B) (A)

(A) (B) (A) (A) (B) (A)

(A) (B) Glucose 6-PO4 Fructose 6-PO4

(i) Shows reaction conditions in which the concentration of reactant, glucose 6-phosphate, is high compared to the concentration of product, fructose 6-phophate. This means

(ii) that the ratio of the product to reactant is small, and RT in Fructose 6- PO4

Glucose 6- PO4

(iii) is large and negative, causing ∆G to be negative despite ∆Go being positive. Thus, the reaction can proceed in the forward direction.

B. Standard free energy change, ∆G: ∆Go is called the standard free energy change

because it is equal to the free energy change, ∆G, under standard conditions that is when reactants and products are kept at 1mol/lit concentration

[B]

= 1 [A]

[B] So, In = 0 [In 1 = 0] [A] And therefore the equation III becomes

∆G = ∆Go + 0

∆Go is predictive only under standard conditions

Under standard conditions, ∆G can be used to predict the direction a reaction proceeds, because under these conditions, ∆Go is equal to ∆G. However, ∆Go cannot predict the

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direction of a reaction under physiological condition, because it is composed solely of constants (R, T and Keq) and is therefore not altered by changes in product or substrate concentrations.

Endergonic processes proceed by couping to exergonic processes The vital processes e.g.1. Synthetic reactions, muscular contraction, nerve impulse. Conduction, and active transport obtain energy by chemical linkage, or coupling, to oxidative reactions.

A Heat D

Chemical C B A+C B+D+ Heat

The conversion of metabolite A to metabolite B occurs with release of free energy. It is coupled to another reaction, in which free energy is required to convert metabolite C to metabolite D. As some of the energy liberated in the degradative reaction is transferred to the synthetic reaction in a form other than heat, the chemical terms “exothermic” and “endothermic” cannot be applied to the these reactions. Rather, the terms exergonic and endergonic are used to indicate that a process is accompanied by loss or gain, respectively, of free energy, regardless of the form of energy involved.

Endergonic A D 1 processes [E] Syntheses

2 Muscular -[E] contraction Nervous 3 Excitation B C 4 Active transport

Free

ene

rgy

E

Exer

goni

c re

actio

ns

Fig. 3. Transfer of free energy from an exergonic to Fig 4. Transfer of energy through a common high An endergonic reaction via a high – energy inter energy compound to energy – requiring (endergonic) mediate compound. processes.

In practice, an endergonic process cannot exist independently but must be a component of a coupled exergonic reactions are termed as catabolism (generally, the breakdown or oxidation of

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the molecules), whereas the synthetic anabolic process constitute metabolism. One possible mechanism of coupling could be envisaged if a common obligatory intermediate (I) took part in both reactions, i.e.

A + C I B + D A. Energy carried by ATP (High energy phosphates play a central role in energy capture and transfer) In order to maintain living processes, all organisms must obtain supplies of free energy from their environment. Heterotrophic organisms obtain free energy by coupling their metabolism to the break down of complex organic molecules in their environment. In all these organisms ATP plays a central role in the transference of free energy from the exergonic to the endergonic processes.

NH2 N N N N9 Adenosine Mg O- O- O-

O- P O P O P O CH2 O C C Phosphate H H H H OH OH Ribose ATP B. Energy carried by ATP ATP consists of a molecule of adenosine to which three phosphate groups are attached. If one phosphate is removed, adenosine diphosphate (ADP) is produced. If two phosphate are removed, adenosine monophosphate (AMP) results.

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1. ∆Go of ATP : The standard free energy of hydrolysis of ATP, ∆Go, is approximately –7300 cal/mol for each of the two terminal phosphate groups. Because of this large, negative ∆Go, ATP is called a high –energy phosphate compound. 2. Very high – energy phosphate compounds: Compounds exist that contain phosphate with energy higher than that of ATP includes ∆Go/

Compounds KJ/mol Kcal/mol Phosphoenolpyruvate -61.9 -14.8 Carbomyl phosphate -51.4 -12.3 1, 3 bisphosphoglycerate -49.3 -11.8 Creatine phophate -43.1 -10.3 3. Low energy phosphate compounds: Other phosphate containing compounds having standard free energy of hydrolysis less than ATP include: ∆Go

Compounds KJ/mol Kcal/mol ADP AMP +Pi -26.7 -6.6 Pyrophosphate -27.6 -6.6 Glucose 1-phosphate -20.9 -5.0 Fructose 6-phosphate -15.9 -3.8 AMP -14.2 -3.4 Glucose 6-phosphate -13.8 -3.3 Glucose 3-phosphate -9.2 -2.2 4. ATP as an intermediate in phosphate transfer: ATP occupies an intermediate position on the bioenergetic scale of phosphate containing compounds. ADP can serve as an acceptor of phosphate groups containing very high energy to form ATP, which, in turn can donate phosphate group in the cell to form lower energy phosphates. There are no enzymes in cells that can transfer phosphate groups directly from very high – energy donors to low – energy acceptors without their first being transferred to ATP. The high free energy change on hydrolysis of ATP is due to relief of charge repulsion of adjacent negatively charged oxygen atoms and to stabilization of the reaction products, especially

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phosphate. As resonance hybrids, other biologically important compounds that are classified as “high – energy compounds” are thiol, amino acid esters involved in protein synthesis, S-adenosyl methionine (active methionine) UDP Glc. (uridine diphosphate glucose) and PRPP (S – phosphoribosyl-1-pyrophosphate). 5. High-energy phosphates are designated by ~(P): To indicate the presence of the high-energy phosphate group, Lipmann introduces the symbol ~(P). The symbol indicates that the group attached to the bond, on transfer to an appropriate acceptor, results in transfer of the large quantity of free energy. Thus, ATP contains two high – energy phosphate groups and ADP contains one, whereas the phosphate in AMP is of the low – energy type, since it is a normal ester link. O- O- O- O- O- Adenosine

O P O ~ P O ~ P O- Adenosine O P O~ P O- ATP O O ADP O O High – energy phosphate act as the “Energy currency of the cell” There are three major sources of ~(P) taking part in energy conservation or energy capture: 1. Oxidative phosphorylation Oxidative phosphorylation is the greatest quantitative sources of ~(P) in aerobic organism. The free energy to drive this process comes from respiratory chain oxidation using molecular O2 within mitochondria. 2. Glycolysis A net formation of two ~(P) results from the formation of lactate from one molecule of glucose generated in two reaction catalyzed by Phosphoglycerate Kinase and Pyruvate Kinase, respectively. Phosphoglycerate Kinase 1, 3 Bisphosphoglycerate 3 –Phosphoglycerate ADP Mg+2 ATP Pyruvate Kinase Phosphoenolpyruvate 3 –Phosphoglycerate ADP Mg+2 ATP

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3. The citric acid cycle One ~(P) is generated directly in the cycle at the succinyl thiokinase step. Succinyl thiokinase ATP Mg+2 ADP + Pi Succinyl-CoA Succinate CoASH Another group of compounds, phosphagens, act as storage forms of high – energy phosphate. These include creatine phosphate, occurring in vertebrate skeletal muscle, heart, spermatozoa and brain and arginine phosphate, occurring in invertebrate muscle.

ATP allows the coupling of thermodynamically unfavorable reactions to favorable one: The first reaction of glycolysis, the phosphorylation of glucose to glucose – 6 phosphate is highly endergonic and cannot proceed as such under physiological condition

(1) Glucose + Pi glucose 6-PO4 + H2O (∆Go/ = +13.8 KJ/mol)

For the reaction to proceed, it must be coupled with another reaction that is more exergonic than the phosphorylation of glucose is endergonic. Such a reaction is the hydrolysis of the terminal phosphate of ATP.

(2) ATP ADP + Pi (∆Go/ = -30.5 KJ/mol)

When (1) and (2) are coupled in a reaction catalyzed by Hexokinase, phosphorylation of glucose readily proceeds in a highly exergonic reaction which under physiological conditions is far from equilibrium and thus irreversible for practical purpose

Hexokinase

Glucose + ATP glucose 6-PO4 + ADP (∆Go/ = -16.7 KJ/mol)

Many “activation” reactions follow this pattern.

Suggested Reading 1. Harpers Biochemistry, Prentice Hall International. 2. Concepts of Biochemistry, L.M. Srivastava. CBS publishers and distributors (2004).