Enzymes

DR. S.CHAKRAVARTY MD

Learning objectives
Define and classify enzymes based on the IUPAC agreement. Give

examples to each class

Classify cofactors and give examples for various types
Discuss the general properties of enzymes and list the important

functions
Define KM, Vmax, Tranistion state and activation energy of enzymes
Discusss the factors affecting enzyme activity
Explain the Michaelis Menten reaction and differentiate it from

lineweaver burk plot. Classify various types of enzyme inhibitions

Definition
Thermolabile biocatalysts which enhances a chemical

reaction without undergoing any chemical change.

Properties :
1.
2.
3.
4.
5.

Specific for a reaction

Does not dictate the direction of a reaction
Increases the rate of a reaction by several thousand
times
Are proteins except ribozymes (RNA)
Lowers the activation energy of a reaction

Enzyme terminology
Simple enzyme made only of proteins
Complex enzyme also called Holoenzyme

Apoenzyme protein part

2. Non-protein part 1.

Prosthetic group with covalent interaction ( usually

metals)
co-enzyme without covalent interaction (vitamins )

Co-enzymes

Group I: take part in reactions transferring

hydrogen atoms or electrons

VITAMIN

COENZYME

FUNCTION

Riboflavin

FMN
FAD

Redoxreactions

Niacin

NAD
NADP+

Redoxreactions

Co-enzymes
Group II: take part in reactions transferring

groups other than hydrogen

Vitamin

Coenzyme

Group
transferred

Function

Thiamine

TPP

Hydroxy ethyl

Transketolase,
oxidative
decarboxylation

Pyridoxine

PLP

Amino or keto

Transamination
reaction

Folic acid

FH4

One carbon
group

One carbon
metabolism

Biotin

Biotin

Carbon dioxide

Carboxylation reaction

Enzymes with metals

Metalloenzyme metal is the indegenous part of the

enzyme itself. Seperation causes disruption

Metal activated enzymes required for activation

but not indegenous to the enzyme.

Metalloenzymes
Metal

Enzyme containing the metal

Zinc

Carbonic anhydrase, Alcohol dehydrogenase,

Iron

Catalase, Peroxidase, Cytochrome oxidase

Xanthine oxidase

Magnesium

Hexokinase, Enolase,Glucose-6-phosphatase
Phospho fructo kinase

Manganese

Hexokinase, Enolase

Copper

Tyrosinase, Lysyl oxidase

Classification of enzymes: based on function

1.

Oxidoreductases

2. Transferases
3. Hydrolases
4. Lyases
5. Isomerases
6. Ligases

1.Oxidoreductases
Involved in oxidation reduction reactions: transfer hydrogen from one

molecule to other
a. Oxidases transfer hydrogen from substrate to oxygen to form water .
cytochrome oxidase
2 AH2 + O2 -------- A + 2H2O
b. Dehydrogenases hydrogen transferred to oxygen indirectly through

hydrogen carriers .
NAD dependent pyruvate dehydrogenase
FAD dependent succinate dehydrogenase

c.

Hydroperoxidase use hydrogen peroxide as substrate to form water

Ex: catalase, peroxidase
2H2O2 ---------- 2H2O +O2

d. Oxygenases -

Monooxygenase single atom of oxygen (hydroxylase)

Ex: Phenylalanine hydroxylase
A-H+O2+ZH2 ---------- A-OH +H2O +Z
Dioxygenase incorporate both oxygen atoms
Ex: Homogentisic acid dioxygenase
A+O2 --------- AO2

2.Transferases
Transfer of groups other than hydrogen:

a.

Aminotransferases : transfer of amino groups

Ex: Aspartate transaminase , Alanine transaminase

b. Acyl transferases : transfer of acyl groups which requires co-enzyme A

(Vitamin B5 (Pantothenate) )
Ex: choline acetylase
C. Methyl transferase: transfer of methyl groups

Ex: Homocysteine methyl transferase

D. Phosphotransferase (kinases) transfer of phosphate groups.

Ex : Hexokinse

3.Hydrolases
Clevage of the substrates by addition of water:
A. Hydrolysis of carbohydrates maltase, lactase,

sucrase etc.
B. Hydrolysis of triglycerides lipase
C. Hydrolysis of proteins pepsin, trypsin, etc
D. Phosphatases phosphodiesterase and glucose -6-

phosphatase.

4.Lyases
Cleavage of substartes or removal of groups by mechanism other

than addition of water.

A. Decarboxylases removal of carbon dioxide from substrates

(require B6)
Ex : histidine decarboxylase
B. Phosphorylases clevage of substrates by addition of

phosphoric acid
Ex : Glycogen phosphorylase

5.Isomerases
Catalyze intramolecular rearrangement, catalyze

conversions between optical, positional and geometric

isomers.
A. Aldose ketose isomerase
B. Epimerase
C. Racemase interconversion of D and L forms
D. Mutase transfer of chemical group from one

position to another in th same molecule.

Ex: glucose -6-po4 -- glucose-1-po4

6.Ligases
Condensation of two molecules to form one molecule

using energy in the form of ATP.

A. DNA ligase
B. Synthetase/synthase
Glutamine synthase

Glutamic acid +ammonia -------------- Glutamine

ATP

Catalysis occurs at the active site

Features of active site

Occupies only a small portion of the whole enzyme
Situated in a crevice or cleft of the enzyme
Possesses a substrate binding site & a Catalytic site
The substrate binds at the active site by weak noncovalent

bonds
The amino acids usually found at the active site are serine,

lysine, histidine, arginine, cysteine

Theories explaining the binding of substrate to the enzyme

Fischers template theory (lock and key model of

enzyme attachment :
According to this theory the active site of the enzyme is

rigid. only a specific substrate complementary to the active

site fits to it just as a key fits to its proper lock

Koshlands induced fit theory:

According to this theory the active site is not rigid & pre-

shaped.
The interaction of the substrate with the enzyme induces a

conformational change at the active site so that proper

alignment of the catalytic residues occur & the substrate fits
in the active site

Fischers template theory

+
E

ES
Complex

Koshlands Induced Fit Theory

+
E

ES
E
Complex

Mechanism of action
Lowering of activation energy
Activation energy is defined as the energy required
to convert all molecules of a reacting substance from
ground state to transition state
Higher the activation energy ,slower the reaction &
vice versa

Activation energy

Mechanism of action

A*

Reactant
[ground state]

[Transition state]

Activation energy (Ea)

B
Product

Mechanism of enzyme activity

1.

Catalysis by proximity

2. Acid-base catalysis
3. Catalysis by strain
4. Covalent catalysis

Catalysis by proximity
High substrate concentration more frequent

encounter and greater will be the rate.

Enzymes bind substrate at active site high local

substrate concentration
Orientation of molecules for better bonding

Acid-base catalysis
The amino acids at the active site contribute to catalysis by

acting as acids or bases

Histidine is often the residue involved in these acid/base

reactions, since it has a pKa close to neutral pH and can

therefore both accept and donate protons.

Catalysis by strain
Enzymes bind to their substrates in a conformation

slightly unfavorable for the bond present in the

molecule which will undergo clevage.
Resulting strain stretches the bond and breaks it.

Covalent catalysis
Involves formation of a covalent bond between the

enzyme and one or more substrates

Modified enzyme becomes a reactant
Creates new reaction pathway whose activation

energy is lower
Serine, histidine, cysteine are involved.

Enzyme kinetics
Quantitative measurement of rates of enzyme catalyzed

reaction and the study of factors that affect the rate of reaction.
A+B

P+Q

The term substrate and products in the above are arbitrary in a

reaction because the reaction can take place in both directions.

The direction of the reaction is dictated by the

thermodynamics (change in free energy or delta G) of the

reaction to which it favours.

Bioenergetics
Also called biochemical thermodynamics

Study of the energy changes accompanying chemical

reactions

Describes the transfer and utilization of energy in

biologic systems

Laws of Thermodynamics:
First law of thermodynamics: The total energy of a

system, including its surroundings, remains constant

Derivation:

H
W
Enthalpy
or Heat content

Q
Heat
absorbed

Work
done

Second Law of Thermodynamics

The total entropy of a system must increase if a

process is to occur spontaneously

Entropy:
# Extent of disorder or randomness of the System
# Maximum in a system as it approaches true equilibrium

Q
Heat

= T S
Temp Entropy

Direction and equilibrium of a reaction

If

USMLE !

G = negative
i.e., free energy of product is less than free energy of
substrates, the direction of the reaction is from left to
right.
these reactions are said to be spontaneous

If

G = positive
i.e., free energy of the product is greater than the free
energy of the substrates, the direction of the reaction is
from right to left favoring substrate formation.

If

G =0, then the reaction is in equilibrium.

Actual free energy change

Delta G is negative

Spontaneous and Exergonic

Delta G is positive

Non-spontaneous and Endergonic

Exergonic and Endergonic reactions

-Coupled
USMLE !
G of two consecutive reactions are additive .
As long as the sum of the Gs of the individual reactions is

negative, the pathway can potentially proceed to

completion even if some of the individual component
reactions of the pathway have a positive G .
Glucose + Pi glucose 6-P + H2O G = +3.3. kcal/mol
ATP + H2O ADP + Pi

G = -7.3 kcal/mol

Glucose + ATP Glucose 6-P + ADP

G = -4.0 kcal/mol

Endergonic process proceed by coupling to exergonic process

The conversion of
metabolite A to metabolite
B occurs with release of
free energy
Free energy is required to
convert metabolite C to
metabolite D

An endergonic process cannot exist independently but must be

a component of a coupled exergonic-endergonic system
where the overall net change is

Factors affecting the rate of enzyme catalyzed reaction

1. Enzyme concentration
2. Substrate concentration
3. Temperature
4. pH
5. Product concentration
6. Presence of activators & Inhibitors

1. Effect of enzyme concentration [E]

on velocity of reaction [v]

Enzyme concentration

2. Substrate concentration
As the substrate concentration is increased the velocity also
correspondingly increased in the initial phases but the curve
flattens afterwards.
A rectangular hyperbola is obtained

Vmax

Vmax

Km

Substrate concentration

Substrate concentration on the reaction rate :

First order reaction
At low substrate concentration the
velocity of the reaction is directly
proportional to the substrate
concentration. This is first order
reaction
Zero order reactions
At high concentration the velocity
of the reaction is independent of
substrate concentration. This is
zero order reaction

3. Effect of temperature
The velocity of enzyme

reaction increases when

the temperature is
increased reaches a
maximum & then falls
The temperature at which

maximum amount of
substrate is converted to
the product per unit time
is called the optimum
temperature

Effect of temperature
Increase in velocity of the reaction

Increase in temperature results in high activation

energy of the molecules & more molecular collision
& interaction for the reaction to proceed faster
Decrease in velocity of the reaction

When the temperature is increased more than the

optimum temperature Denaturation of the enzyme
occurs

4. Effect of pH
The pH decides the

charge on the amino acid

residues at the active site.
The net charge on the
enzyme would influence
the substrate binding &
catalytic activity

5. Effect of product concentration

The accumulation of reaction products generally

decreases the enzyme velocity.
The products combine with the active site of the
enzyme & form a loose complex & thus inhibit the
enzyme activity.

6.Factors affecting enzyme activity

Activators: Substances that increases the enzyme activity.
Eg : Zinc activates carbonic anhydrase
NAD+ activates LDH
Inhibitors: substances that decreases the enzyme activity
Eg : Fluoride inhibits Enolase.
Cyanide inhibits cytochrome oxidase.

Specificity of enzyme activity

Sterospecificity
Reaction specificity
Substarte specificity Absolute

Relative
Broad

Reaction specificity
The same substrate can undergo different type of reaction.
Each reaction is catalysed by a separate enzyme

se
a
l
xy
o
rb
Ca

Oxalo acetate

PDH

Pyruvate
Tr
an

Malic enzyme

Lactate

LDH

Malate

sa
m
in

Acetyl coA

as
e

Alanine

Substrate specificity
Absolute specificity : Enzymes that can act only on one substrate & can

catalyse only one reaction

Glucokinase

Eg : Glucose

Glucose-6-phosphate

Group specificity : Carboxy peptidase & amino peptidase are

exopeptidase which hydrolyse peptide bond in the vicinity of free

COOH or NH2 groups respectively
Broad specificity : Hexokinase acts on Glucose, Fructose, Mannose etc

Michaelis menten graph

X- axis substrate concentration
Y-axis velocity of the reaction

(V)
Vmax

= maximum velocity a
reaction can reach. Directly
proportional to amount of the
enzyme and substrate

Enzyme concentration is kept constant.

As
the
substrate
concentration
increases the velocity increases but
after some conc.. The graph flattens
due to enzyme saturation. The highest
point on the graph is called Vmax.

Km = substrate conc at the half

maximal velocity.

Representation of enzymes at various substrate concentration

Michaelis menten equation

Vi

Vmax [S]
Km + [S]

1) When substrate conc < Km value (point A), Vi will be directly proportional to
substrate conc
2) When substrate conc = Km value(point B), vi wil be half the maximal velocity
3) When substrate conc > Km value(point C), Vi wil be equal to Vmax and
independent of substarte conc on further increases.

What does the Km value say about a reaction?

High Km value means the enzyme has low affinity for the

substrate .
We have to load a large amount of substrate for the reaction to
attain Velocity.
Low Km value means - the enzyme has very high affinity for

substrate.
Even if small amounts of substrates are there the reaction will
attain velocity
Affinity / binding is inversely proportional to Km value.
Binding is good when shapes of both enzyme and substrate match

Hills equation;

Y=ax+b

Conversion of michaelis mentan equation which is hyperbolic to

hills equation which is linear.

Lineweaver Burk Double reciprocal plot

X axis = 1/S
Point of intersection on x

axis = (-1/Km) value

Y axis = 1/V
Point of intersection on Y-

axis(slope) = 1/Vmax
Inverse plot of michaelis menten both S conc and velocity plotted by
taking 1/S and 1/V on x and y axis. This was done to make the equation
linear.

Enzyme inhibition
A substance that can inhibit enzyme activity is called

enzyme inhibitor:
Types :

Competitive inhibition
2. Non-competitive inhibition
3. Uncompetitive inhibition
4. Mixed inhibition
1.

Competitive inhibition
Inhibitor resembles substrate

in shape.
Substrate competes with

catalytic site.
Inhibition is reversible

because the inhibitor forms

non-covalent bonds
Inhibition can be reversed by

increasing substrate conc..

So in competitive

inhibition:
Km is increased
Vmax remains unaltered

because the number of

enzymes remain the
same.

Competitive inhibition:
Michaelis menten plot :

Lineweaver plot:

Methanol Poisoning
Wood alcohol and antifreeze contain high methanol concentrations
Methanol poisoning causes decreased blood pressure and body

temperature, and an increase in respiratory rate

Methanol + NAD+
Ethanol + NAD+

Alcohol Dehydrogenase
Alcohol Dehydrogenase

Formaldehyde + NADH + H+
Acetaldehyde + NADH + H+

- FOMEPIZOLE IS THE CURE FOR BOTH

Alcohol dehydrogenase has a slightly lower Km for ethanol compared to
methanol
The additional benefit of the ethanol reaction is that acetaldehyde is less
physiologically damaging than formaldehyde, which is toxic to the retina
and can cause permanent blindness if doses are high for prolonged periods
of time
Displaced methanol can be safely excreted by the kidneys

Methotrexate Structural analogue of folate

inhibits folate reductase

Dicoumarol Structurally similar to Vit

Kanticoagulant
Sulfonamide antibiotics structural analogues of

PABA inhibits folate synthesis in bacteria

Non-toxic to humans humans dont synthesize PABA

Non-competitive inhibition:
Inhibitors bind to site other than substrate binding

site.
Covalent bond formation occurs
Loss of enzyme activity.
So Vmax decrease but Km unaltered.

Non-competitive inhibition
Michaelis menten plot:

Lineweaver plot:

Uncompetitive inhibition
Inhibitor binds to the enzyme substrate complex.
Increased Substrate affinity apparently decreases the

Km but Vmax is decreased because the enzyme will

take a long time to separate from the complex.

Uncompetitive inhibition
Lineweaver plot:

Km value decreases
Vmax also decreases

Summary of inhibition:

Enzyme inhibition

Km

Vmax

Competitive inhibition

Increases

Unaltered

Non competitive
inhibition

Unaltered

Decreases

Uncompetitive
inhibition

Decreases

Decreases

Mixed

Increases

Decreases

Enzyme regulation
The process by which cells can turn on, turn off, modulate

the activities of various metabolic pathways

For coordinating various processes as per the physiological

needs of the body.

Every pathway has enzymes which are the rate limiting/ or

key enzymes

Types of enzyme regulation

Coarse regulation Regulating enzyme activity
Fine regulation Regulating enzyme concentration.

Types of enzyme regulation

Coarse
Regulation:

Covalent modification
Allosteric regulation
Feed back inhibition
Compartmentalization

Fine Regulation:
Induction
Repression

Allosteric Regulation
These are the enzymes having one Catalytic site & another

separate site called Allosteric site where the modifier binds &
regulate the enzyme activity.

Catalytic site
(substrate )
Allosteric site
(allosteric compound)

Allosteric Activation

Substrate

Allosteric activator

Enzyme

Enzyme substrate
Complex

Enzyme

Product

Allosteric inhibition

Substrate

Allosteric inhibitor

Enzyme

No enzyme substrate
Complex

Allosteric regulation

Positive modifier (activato

Sigmoid curve
Negative modifier
(inhibitor)
Substrate concentration

Covalent modification
Addition of a group to the enzyme by a covalent
bond or removal of a group by cleaving a covalent
bond
Types

Adenylation - deadenylation
Methylation
Ribosylation
Acetylation
Phosphorylation(become inactive)dephosphorylation(become active)

Feedback inhibition

The process of inhibiting the first step by the final

product, in a series of enzyme catalysed reactions
of a metabolic pathway

e1

E (-)

e2

e3

e4

Compartmentalization
Localisation of
enzyme

Metabolic pathway

Cytoplasm

Glycolysis, fatty acid

synthesis

Mitochondria

Krebs cycle, ETC, Fatty

acid oxidation

Peroxisomes

Long chain fatty acid

oxidation

Both mitochondria & Urea cycle, Heme

cytosol
biosynthesis