Enzyme

Enzymes are commonly proteinaceous biocatalyst which are capable of

catalyzing chemical reactions of biological origin without themselves
undergoing any changes.

Enzymes are proteins (polymer of amino acids).

Recently some RNAs have been discovered to have enzymatic function ----- Called
Ribozyme.
 Chemical nature of enzymes

Simple Protein enzymes Conjugates enzymes

(Wholly made up of protein)
Pepsin, Trypsin, Urease
Holoenzyme

Co-Factors
Apoenzyme Non-protein part of enzyme
Protein part of enzyme

Prosthetic group Metal ions

Co-enzyme
organic compounds and are A number of enzymes require
organic compounds but their
distinguished from other cofactors metal ions for their activity which
association with the apoenzyme is
in that they are tightly bound to form coordination bonds with side
only transient, usually occurring
the apoenzyme chains at the active site and at the
during the course of catalysis.
Example: Haem, FMN, FAD same
Example: NAD, NADP
time form one or more cordination
FMN = Flavin Mononucleotide bonds with the substrate
FAD = Flavin adenosine dinucleotide Example Zn for carboxypeptidase
 Enzymes are Proteins --- not true

Apoenzyme Cofactor /coenzyme

Protein part Non-protein part
Large in size Small In size
Participate in catalytic activity Binds between enzyme and
substrate
Not help in group transfer Helps in group transfer
Thermolabile Not Thermolabile
Prosthetic group Coenzyme

Organic non-protein part attached firmly to an Organic non-protein part attached loosely to an
apoenzyme (protein part) apoenzyme (protein part)

Non-dissociable organic cofactor dissociable organic cofactor

Example Heme Example NAD (necotinamide adenine

dinucleotide), NADP (necotinamide adenine
dinucleotide phosphate)
 Co-factors are vitamins

Enzymes Coenzyme/ Vitamins

Prosthetic gr.
Decarboxylase Thiamine Vit B1 (Thiamine)
pyrophosphate
Dehydrogenase FMN and FAD Vit B2 (Riboflavin)
Trans-aminase Pyridoxal Vit B6 (Pyridoxine)
Phosphate
Dehydrogenase NAD Niacin
Synthetase Co-enzyme Pantothenic acid
 Turn Over Number

The number of moles of substrate converted per minute by one mole of enzyme.

Carbonic anhydrase ----- Fastest enzyme ---- Kcat value = 36 million / minute.
Lysozyme ------- Slowest enzyme ------- 3 million /minute

Catalase = highest turn over number (some books)

 Definition
Isozyme

Physically distinct forms of a single enzyme having same catalytic

activity.

Example: Lactic dehydrogenase ----------- 5 Isozymes LH, LH2, LH3,

LH4 and LH5.

Zymogens
Precursors of enzyme; not active on their own but become active
after conversion.

Ex. Pepsinogen.

Allozyme
They are similar enzymes are produced by different genes

Ex. DNA polymerase.

Q10 value= Rate of reaction increases from 2 to 3 times for 10C
increase.

Ki = Dissociation constant of enzyme-inhibitor complex.

Low Ki = High enzyme activity.

High Ki = Low enzyme activity.
 Metal activators of enzyme

Enzyme Name Metal activator

Phosphatase Mg 2+
Aminopeptidase Mn 2+
Carbonic anhydrase/ Carboxypeptidase Zn2+
Kinase / enolase Mg 2+
Cytochrome Oxydase Fe 2+ and Cu 2+
Amylase Cl-
DNA polymerase Mg 2+
Urease Ni
 Discovery:

1. The existence of biological catalysts was predicted by the Swedish chemist, Jon
Jakob Berzelius, in 1835.
2. Eduard Buchner = discovered enzyme, Isolated 1st enzyme Zymase , awarded
Nobel prize
3. Kuhne (1878) coined the word enzyme.
4. J.B. Sumner : isolated enzyme urease from jack bean meal in 1926, and his
discovery that enzymes are proteins.
5. Northrop (1930-35) isolated pepsin, trypsin and chymotrypsin
6. James B. Sumner, John H. Northrop and Wendell M. Stanley were awarded Nobel
Prize in Chemistry in 1947 for their work on isolating enzymes in pure crystalline
forms for the first time.
 Active site

An area of the enzyme which is capable of attaching and holding particular

substrate molecule by its specific charge, size, and shape, so as to allow the
chemical change.

Active site is made up of R-group of selected amino acids.

 Activation Energy

The energy required to convert all molecules in one mole of a reacting substance
from the ground state to the transition state.

Over all energy change to

reaction
Classification of Enzymes

1. Oxidoreductases/dehydrogenases

2. Transferases

3. Hydrolases:

4. Lyases:

5. Isomerases:

6. Ligases:
 Classification of Enzymes

Classes of enzymes Reaction Examples

1. Enzymes which catalyse Malic dehydrogenase,
Oxidoreductases/dehydrogenases oxidoreduction between two substrates Urease
: S and S’ Oxidase

S reduced + S’ oxidised  S oxidised + S’ reduced.

 Classification of Enzymes

Classes of enzymes Reaction Examples

Enzymes catalysing a transfer of a group, Hexokinase

2. Transferases G (other than hydrogen) between a pair Transaldolase
of substrate S and S’

S - G + S’  S + S’ - G
 Classification of Enzymes

Classes of enzymes Reaction Examples

3. Hydrolases: Enzymes catalysing hydrolysis of ester, ether, Esterase

peptide, glycosidic, C-C, C-halide or P-N Peptidase
bonds.
 Classification of Enzymes

Classes of enzymes Reaction Examples

Enzymes that catalyse removal of groups Carboxylase

4. Lyases: from substrates by mechanisms other Aldolase
than hydrolysis leaving double bonds. Enolase
 Classification of Enzymes

Classes of enzymes Reaction Examples

Includes all enzymes catalysing Isomerase

5. Isomerases: inter-conversion of optical, Epimrase
geometric or positional isomers.
 Classification of Enzymes

Classes of enzymes Reaction Examples

Enzymes catalysing the linking together Succinic thiokinase

6. Ligases: of 2 compounds, e.g., enzymes which Glutamine synthetase
catalyse joining of C-O, C-S, C-N, P-O etc.
bonds.
 Enzyme kinetics

Km or Michaelis-Menten constant: This constant indicates the substrate concentration

at which an enzyme catalysed reaction attains half its maximum velocity (½ Vmax)

Vmax : The reaction ultimately reaches a maximum velocity (Vmax) which is not
exceeded by any further rise in concentration of the substrate.
 Mechanism of Enzyme action

1. Lock and Key Theory . …………… Emil Fischer.

2. Induced Fit Theory …………………. Daniel E. Koshland

 Lock and Key Theory

E
Enzyme
+

Substrate
E E
Enzyme
+

Products
Enzyme-
Substrate
complex

1. Each enzyme (E) has a substrate (S) binding site in its molecule so that a highly reactive enzyme-
substrate complex (ES) is produced.
2. This complex is short-lived and dissociates into its product(s) P.
3. The unchanged enzyme with an intermediate formation of the enzyme-product complex (EP).
4. Active site is rigid.
 Induced Fit Theory
Buttressing Group c
C d
b
D
a e Substrate
A E
Enzyme

Catalytic Group

C
cd
D
b

a e E Enzyme-Substrate complex
A

Buttressing Group
C

D
Products
A E
Enzyme

Catalytic Group
 Difference between Lock and Key Theory and Induced Fit Theory

Lock and Key Theory Induced Fit Theory

Active site is single entity Active site is made up of two components

There is not separate catalytic A separate catalytic group is visualized.

group

The buttressing group of Active site

Active site is static.
undergoes conformational change.

Development of transition Development of transition

state is not considered. state is considered.
 Mechanism of Enzyme Action

The catalytic cycle of an enzyme action can be described in the following steps:
1. First, the substrate binds to the active site of the enzyme, fitting into the active
site.
2. The binding of the substrate induces the enzyme to alter its shape, fitting more
tightly around the substrate.
3. The active site of the enzyme, now in close proximity of the substrate breaks the
chemical bonds of the substrate and the new enzyme- product complex is formed.
4. The enzyme releases the products of the reaction and the free enzyme is ready to
bind to another molecule of the substrate and run through the catalytic cycle once
again.
 Allosterism
The property of the enzyme by which, when an effector molecule binds with
enzyme, the conformation of the active site of the enzyme is changed favourably
or unfavourably to the substrate binding, is known as allosterism.

Allosteric Enzymes
The enzymes which possess a site in addition to their active site to which an
agent may be bound to modify (activate or inhibit) the enzymatic activity.

Active site = site for binding substrate molecules

Allosteric site = Controls the

conformation of active site
E

(activate or inhibit)
Allosteric Modulation and Feed Back Mechanism

Glucose
Hexokinas

G6P

Feed back
mechanism
 Factors affecting enzyme activity

Temperature Increase rate due to greater kinetic movement of

molecules

Decrease rate due to

denaturation of
enzyme

Velocity of reaction
Actual rate of reaction
balance between the
other two opposing
influence.

Temperature/ C

1. Enzymes generally function in a narrow range of temperature.

2. Each enzyme shows its highest activity at a particular temperature called the optimum temperature.
3. Activity declines both below and above the optimum value.
4. Low temperature preserves the enzyme in a temporarily inactive state whereas high temperature destroys
enzymatic activity because proteins are denatured by heat.
 Factors affecting enzyme activity
Salivary
pH Pepsin Amylase Trypsin
pH 6.8 pH 8.5
pH 2.5

Velocity of reaction

pH

1. Enzymes generally function in a narrow range of pH.

2. Each enzyme shows its highest activity at a particular pH called the optimum pH.
3. Activity declines both below and above the optimum value.
 Concentration of Substrate

1. With the increase in substrate concentration, the velocity of the enzymatic reaction rises at first.

2. The reaction ultimately reaches a maximum velocity (Vmax) which is not exceeded by any further rise
in concentration of the substrate.

3. Why velocity of reaction not exceeded further rise of substrate concentration?

------------This is because the enzyme molecules are fewer than the substrate molecules and after saturation
of these molecules, there are no free enzyme molecules to bind with the additional substrate molecules.
Enzymes
1. What is enzyme? State the properties of enzymes.
2. Differentiate between:
a) Enzymes and inorganic catalysts.
b) Coenzyme and prosthetic groups.
c) Lock and key mechanism and Induced fit model.
d) Apoenzyme and cofactor.
e) Primary metabolites and secondary metabolites.
f) Anabolism and catabolism.
g) Competitive inhibition and allosteric inhibition.
3. Define with examples: Allosterism, Allosteric enzyme, Activation energy, Active site,
Abzyme, Zymogen, Isozyme, Acivator, Inhibior, Allosteric modulator, Irreversible
inhibition, Km and Vmax
4. What is competitive inhibition? Explain with an example.
5. What is non-competitive inhibitor? Explain with an example.
6. Classify enzymes according to IUB with examples.
7. Explain effect of heat and pH on enzyme action.
8. Explain Induce fit model with suitable diagram.
9. Explain : E + S ES complex  E + P
 The Enzyme Inhibition

The activity of an enzyme is also sensitive to the presence of specific chemicals that
bind to the enzyme. When the binding of the chemical shuts off enzyme activity, the
process is called inhibition and the chemical is called an inhibitor.

Types of enzyme Inhibition:

1. Reversible Inhibition.
a) Competitive Inhibition.
b) Non-competitive Inhibition
2. Irreversible inhibition.
 Competitive Inhibition.
1. When the inhibitor closely resembles the substrate in its molecular structure and inhibits the
activity of the enzyme, it is known as competitive inhibitor.
2. Due to its close structural similarity with the substrate, the inhibitor competes with the
substrate for the substrate binding site of the enzyme.
3. Consequently, the substrate cannot bind and as a result, the enzyme action declines,
4. e.g., inhibition of succinic dehydrogenase by malonate which closely resembles the substrate
succinate in structure. Such competitive inhibitors are often used in the control of bacterial
pathogens.
 Competitive Inhibition.

Velocity of reaction

½ V max

Substrate concentration
Km
 Non-Competitive Inhibition.

A non-competitive Inhibitor does not resemble the substrate in structure.

It binds to the enzyme at some site other than the active site or binding site of
substrate and renders it inactive by altering the enzyme conformation.

Velocity of reaction

½ V max

Substrate concentration
Km

Example: Cyanides block action of some enzymes by combining with iron which may
be present in prosthetic group.