Chemical reaction in cells are often coupled

Glucose + Pi Glucose-6-phosphate

(requires input of energy, endergonic)

ATP ADP + Pi

(releases energy, exergonic)

Reaction 1-

Reaction 2-

Reaction 1 + Reaction 2-Glucose + ATP Glucose-6-phosphate + ADP

(releases energy, exergonic)

Biochemical reactions are generally parts of metabolic pathways

Each step may not be energetically favorable, but the overall pathway must be exergonic

Biochemical reactions are generally parts of metabolic pathways

Each step may not be energetically favorable, but the overall pathway must be exergonic

Most small molecules are synthesized using such pathways

Enzymes are tightly regulated

Cells respond to their environment through the enzymes

Isoleucine starvation

Living organisms are broadly divided into two categories

Eukaryotes(contain a nucleus)

and

Bacteria(no nucleus)

~50 m

Three domains of living organisms

Bacteria lack a nuclear membrane and are relatively simple

Cell envelope: Contains cell walland membrane(s)

Gram positive:one membrane

Eukaryotic cells are much more complex

A hydrogen bond is formed through electrical interaction between an atom covalently bonded with a hydrogen and which is more electronegative than hydrogen, and another atom that has a partial negative charge.

Hydrogen bonds can also form between other functional groups

Despite their diversity, all proteins are made from 20 commonmonomeric units called amino acids

Amino acids are carboxylic acids CO

OH

H

2

and contain an amino group

(Uncharged)

(Uncharged)

Phe

UV absorption

Cysteine can exist in two forms

Oxidation

Reduction

There is delocalization of electrons along the peptide bond

This creates, 1. An electric dipole moment within the peptide unit

O

N

Cα H

Cα

Trans

Cα

N

O H

Cα

Cis

2. Prevents rotation around the amide linkage

3. Makes each Cα-CO-NH-Cα flat

Proline does not have a strong preference for the trans configuration

4:1

The planar units around N-Cα and Cα-C (=O) bonds

Ramachandran Plot

Only certain combinations of angles are “allowed”

Regular structures in proteins α-helix

=On•••H-Nn+4-

1

2

3 4

5

67

8

9

10

R-groups are on the outside of the helix

Myohemerythrin

Helix bundle

-sheet

R-groupsabove andbelow

-strand

-sheet

R-groupsabove andbelow

-strand

Thioredoxin

Overall folding of the protein

The presence of disulfides reduces the number of alternate structures

Pot

enti

al e

nerg

yP

oten

tial

ene

rgy

Most enzymes are proteins

The exceptions are a handful RNA molecules that can act as enzymes

They are called ribozymes

Consequently, the rate of an enzymatic reaction is highestwhen the enzyme is “saturated”

[E] is constant

Rat

e

Maximumvelocity

Enzymes are often aided by metals

Enzymes that use metals (other than Na+ and K+) are calledmetalloenzymes

…...and by coenzymes

Energetics of enzyme action

S (substrate) P (product)E (enzyme)

Keq =[P][S]

Keq is equillibrium constant

(Molar concentrations)

Keq =[P][S]

Keq is equillibrium constant

ΔG' 0 =−RT lnKeq'

G is Gibbs free energy, G’0 is change in Gibbs free energyunder the following conditions: Temp 37°C (298 °K), pH 7.0,1M reactantsR is Gas constant (8.3 J/mol or 2 Cal/mol)

All but the last condition is always satisfiedWe may drop the superscript “’” and “0”, but it is always assumedto be present

E+S↔ ES→ E +Pk1

k-1

k2

Michaelis-Menten kinetics

Assumptions: 1. There is a constant amount of enzyme-substrate complex during the course of the reaction

Also known as- Steady-state kinetics

Constant

E+S↔ ES→ E +Pk1

k-1

k2

Michaelis-Menten kinetics

Assumptions: 2. There is no reverse reactioni.e. the reaction is irreversible

Also known as- Steady-state kinetics

If you multiply both sides of the equation with k2 then...

k2[ES]=k2[ET ][S]Km +[S]

k2[ES] = v (velocity of the reaction)k2[ET] = k2[ETS] = vmax (the maximum velocity of the reaction)

∴ v=vmax[S]Km +[S]

Michaelis-Menten Equation

Turnover number (kcat) is defined as

kcat = vmax/[ET]

This defines the rate of catalysis per enzyme molecule

Turnover number (kcat) is defined as

kcat = vmax/[ET]

This defines the rate of catalysis per enzyme molecule

Specificity constant is defined as

kcat/Km

Types of enzyme inhibition

1. Competitive inhibition

Types of enzyme inhibition2. Non-competitive inhibition

Types of enzyme inhibition3. Irreversible inhibition

Other ways of regulating enzymes

1. Allosteric control

An enzyme is said to be regulated allosterically when a molecule binds to a site other than the active site and modulates enzyme activity

2. Covalent modifications

Enzymes are often (reversibly) covalently modified to changethere activity

3. Proteolytic cleavage

Some enzymes are activated by proteolytic cleavage

Schematic view of allosteric activation

Example of activation: Hemoglobin

Fra

ctio

n of

bin

ding

sit

es

occ

upie

d

Sigmoidal shape ischaracteristic ofpositive allosterism

Negative regulation of biochemical pathwaysis often allosteric

Threonine dehydratase is negatively regulated by L-isoleucine

Feedback Inhibition

Covalent Modifications of Proteins

Perhaps the most important of these is phosphorylation

O P O

O-

O-

OH + ADP+ ATP

Kinase

ATP ADP

Kinase

PhosphataseActive Inactive

Proteolytic cleavage of proteins

Purification of proteins

1. Size Exclusion chromatography (aka Gel Filtration)Separation of proteins by size

Larger (bigger) proteins are “excluded” and go through faster

Smaller proteins are retained by the beads and go through slower

Ion-exchange chromatography

In a cation exchanger, thepositively charged proteins will tend to stick to the column and move slowly through the column.In contrast, negatively charged proteins will move faster through the column

Opposite is true for the anion exchangers.

2. Analysis of the subunit composition of the protein

A. Determination of MW by Gel filtration Chromatography

RelativeElutionVolume

+

++

++

++

++

+

Log MW

Proteins with known MW

Larger proteins elute first (in smaller elution volume)

B. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

S

O

O

-O O (CH2)11 CH3Na+

Sodium dodecyl sulfate

Polyacrylamidematrix

Breaks disulfide bridges

HOH2C CH2SH

2-mercaptoethanol

1 2 3 4 5 6

Larger proteins elute later (migrate less distance on the gel)

3. Amino acid composition of the protein

Heat (>105°C)24 Hr

Which amino acids are missing?

Tryptophan (May be accounted for by UV absorbance)

Gln Glu

Asn Asp

O

NH2

Acid HydrolysisO

O-

Hence,Amt. of Glu (by acid hydrolysis) = Amt. of Glu + Amt. of GlnAmt. of Asp (by acid hydrolysis) = Amt. of Asp + Amt. of Asn

4. Amino acid sequence of protein

Edman degradation