INTRODUCTION TO ENZYMOLOGY

Enzymes are protein specialized to catalyse biological reactions (Biocatalyse):

Increase the speed of reactions

Thumbers do not changes in the reaction.

Very important to life as life depends on biological reactions (example

digestion).

Any change in a single enzyme can have very harmful effects.

Very specific

Enzymes (E) act on certain substances which are known as substrate(s).

They form an Enzyme-Substance Complex (ES) which is broken or changed
to give the Product (P).
E+S '

ES

E+P

Substance on which an
enzyme acts

The final substance that

that is produced.

Cofactor: Some enzymes depend for activity only their structure and proteins,
while others require one or more non-protein component. This is known as the
Cofactor. Cofactor may be metal coin or an organic molecule called Coenzyme. Some
enzymes require both:
Apoenzyme +
(Catalytically
inactive)

Cofactor

Haloenzyme
active (enzyme)

Cofactors are generally stable to heat while the enzyme protein loses its activity on
heating.

Metal as cofactor
e.g.

Alcohol dehydrogenase

Zn ++

Kinases (phosphotransformer)

Mg++

Cytochromes

Fe++ or Fe+++

Cytochrome oxidase

Ge++

Coenzymes

Papidoxal phosphate

Amino transferase

NAD+

in H. transfer

NADP

FAD

FMN

COQ

COA

Amyl group transfer

Most of the coenzymes contain as part of its structure and molecule of one or
another of the vitamins. Coenzyme usually function as intermediate carriers of
functional groups of specific atoms or of electrons.

Where the coenzyme is tightly bond to the enzyme molecule, it is usually called a
prosthetic group e.g. biocyte group of acetyl CoA carboxylases.
In some cases the co-enzyme is only loosely bound to the enzyme protein.

The enzyme combined with the substrate at a specific site known as the active site:

Active site

Inhibitors some substances combined with the enzyme and reduce its activity.
These are known as inhibitors.
2

Activators. Others they combine with the enzyme or increase its activity, these are
known as activators.

Activation: Process by which the enzyme molecule becomes more active.

Brief History of Development of Enzymology

1833
Payen and Persoz found that alcohol precipitate of malt extract contained a
substance that was thermolalite and that converted starch into stager. They
called it disease (now known as amylase) of its power of separating soluble
dextrin from the insoluble starch grains.
1894
Duclaux proposed that to name an enzyme the name of the substance on which
the enzyme acted. This is still used (a few enzymes contain in as the last 2
letters especially those of the digestive treat). (Now IUB system of
nomenclature).
1860
Lonis Pasteur recognized that for mutation.
1878
W. Kuhine introduced the same enzyme
1897
Edward Buchners succeeded in extracting from yeast cells the enzyme
catalyzing alcohol for mutation.
1922-1928
Willstalter and his colleagues carried out studies to purify enzyme.

1926
J.B. Summer purified increase from extracts of Jack bear. He crystallized the
increase.
1948
Crystallize proteolytic enzymes were isolated by Northrop and his colleagues.
Todate
Over 2000 enzymes are known and ~ 200 have been crystallized.
Recent world has known light on structure of enzymes, mechanism of enzyme
reactions, control of enzyme synthesis. Complete detailed 3D structure of enzyme:

Classification of Proteins (briefly)

e.g. Enzymes are proteins:

Exist as Zwitterious isoelectric pH. Above this pH newly charged, below and
newly charged
o can move in an electric field (useful in separation methods).

Can be denatured loss of structure and shape: loss of function. pH, heat,
radiations, high salt concentration, detergents, 8M urea/-mcorpts ethanol metal
ions.

Mol nt: ranging from 1000 m several thousand.

Have buffering capacity (acid base property)

Absorption spectra One toTmp, Tye, Phe absorb light in the visible range (in
the uV) at 280.

Enzyme reduce the energy of activation

Speed up the reaction.
Reaction A ' B goes the rough a transition

The rate of the formed reaction depends on kan temp and on the difference in free
energy between A and the transition state.

Enzyme u reactions by v G+, the activation barrier. The combination of E+S

creates a new reaction pathway whose transition state energy is lower than reaction
with E.

Enzymes are highly specific

-

Absolute specificity

Reaction specificity

Sterio specificity

Group (class specificity)

e.g. substitution (bacteria) can break all peptide bonds

Types

C106

N
IH

Agr Lys
Chymotrypsin
Phe Thy Trp
Peps

Ale Tyr Top

DNA polymerase :

However, enzymes vary in the degree of specificity

very specific

o Group specificity relatively non specific for a group of diameter

compounds

e.g. alkaline phosphate hydrolyses many different esters of phosphoric acid

carboxypeptidase catalysis the hydrolysis of c-terminal peptide bond of peptides.

Since most proteins contain Tyr residue measurement of light absorption at 280 nm
in the spectrophotometer is an extremely rapid and convenient method for certain
proteins in solution.

Optically active (contains only C aa)

Free N-terminal that can react with ninhydrin to form a colored derivative.

Binver reaction is given by peptide and proteins but not by a.a. P + Cm ++ alleli,
Purple Cm++ - peptide complex.

Reduction of disulphate (by -mercapto ethanol).

Enzyme Specificity
1. Enzymes act on either one substance or a group of closely related substrates i.e.
they are specific.
2. Very important characteristic of all C.
3. Most important biological phenomenon
determine velocity change
4. E
determine which substance undergo change
E help in organized to organize the metabolic pathway
1. Organization by specificity
5. Degree of specificity varies with different E . Some E only only one one 5 and
catalyse only one reaction.
1. Others act on a small No. of closely related substrates, carrying out the same
reaction in all cases (A common chemical structure can be recognized in all
the substrates on which the E acts. For less specific E this group is a male
molecule e.g. aldehydic or ester.
Investigation of Specificity: For satisfactory investigation:
1. Pure E (preferably recrustallized several times)
2. Free of other E which act on similar S
3. Pure S and free of other S on which E may act.
4. 2 opt active isomers must be tested separately (some of them may inhibit
the reaction).
5. 1st use most readly attached biological 5 then examine other S.
6. For each S work out Kim and Vmax (using time-weaker and bul plot)

7. For ionizing S Kim and Vmax must be obtained for a wide range of pH.
Obtain opt pH curve.
8. Study effect of competitive I to investigate the influence of structure.
9. Use low conc. of I to avoid reactions due to traces of other E.
10. Make small chemical modifications in E and study effect of this on the
activity and reactivity.
11. Formulate the minimal structure necessary for combination and for reaction.
Types of Specificity
The specificity is due to substrate binding site which has on the enzyme surface.
The specificity is also due to the specific arrangement of a.a. that participate in the
bond making and bond breaking phase of catalysis:
Models to explain substrate specificity
1. Lock and key model (Fischer)
2. Induced fit model

1.

Absolute Specificity
The E com act only on 1 specific substrate:
Glucokinase
Glucose

Glucose-6-phosphate
ATP

ADP

Asparate
1. Asparate

Formulate + NHm

Coo-

Coo
CH

HC
Coo-

CH2 +
H+
H C NH2
Coo-

+ NHu

(only on L isomer)
Lactate dehydrogenase
Pyruvate

L-lactate
NADH

2.

NAD+

Group Specificity

Bound specificity

E act an a group of related S

The substrates have a common group on which the E acts.

e.g. esteroses can act on esteri proteases ------ protein.

3.

Stereo Specificity
-

E can act only on one type of optical isomer or geometrical isomer

(usually absolute specificity).
e.g. chymotrypsin acts on -isomers of acetyle-L-tryptophenamide, AcetyleL-tysosinamide, and N=catamyl-L-ltryptophin.

Aim etc., but not E the D-isomer. In fact the D-isomer acts as an inhibitor.

If the S is symmetric but the product is asymmetric, the E gives only 1

isomers of the product e.g.
LDH
Pyr

L-Lactate
Other dehydration

Pyr

D-Lactate
D-LDH
10

GDH
-ketoglu + NH3
-

L-glutamate

The antipode may act as a inhibitor of the E.

Cis-trans Isomerism
-

Often the enzyme acts on only 1 form of genometric isomers cis or trans
H

H
or

C
H

e.g. Furmarate hydrotase

CooH

CooH

CH

CH

for hydrolase

HO C

CooH

CooH

Furmarate

Molate

Highly specific
-

Dehydrogenase

e.g.

alcohol dH.
aldehyde DH
Acyl-CoA du

Highly specific
Only 1 pair of S is known (i.e. H-donor and H-acceptor)
-

Glucose oxidate

Highly specific acts only on D-glucose

on D maumase D-Altrose
D Gal, only very slow rate.

Flaro protein
11

Amino acid oxidase

Kinases
Creatine kinase
Creatine

Phospho-c reactive
ATC

ADP
HR

Hexoses

Hexose-6-P
ATP

Estors

ADP

e.g. Liposes
(acetylcholine ester)
CH3

Co

CH2

CH2

CH3
CH3
CH3

Acetylcholine
Hydrolyse choline and non-choline esters.
Phosphatase
Alkaline (P)
Glycosol-1- (P)

Glycosol + (P)

Glycosol-2- (P)
P~P

P+P

Nucleases

Break nucleic acid

Glycosidase
Peptidase
Increase

- First considered to have absolute specificity

- But now know that it may act on urea derivatives.

Highly Specific
May lyases

e.g. Pyr decarboxylase

Furmate hydrolase
L malate

'

Furmate
12

Emolase

L- phosphor D glycerate
Emolase
PEP

Isomerase
Aldolase

1- epimerase
- Sugar ' - sugars

13

ENZYME STRUCTURE

Protein in nature

Four levels of protein structure.

o Primary
o Secondary
o Tertiary
o Quartering

Classification of proteins
o Basis of information
o Catalytic enzyme
o Transport function
Albumin
o Protective (immunological)
Antibodies
o Coagulation
Thrombin
o Mechanical support
Collagen, Kerantin
o Contractile function
Action and lyosin in muscle
o Enzyme inhibitors
o Storage
Aproferritin  Ferritin
Myoglobin

14

o On the basis of composition

- Simple proteins
- Conjugated proteins

Glycoproteins

Hemoproteins

Metaloproteins

Lipoproteins

Phosphoproteins

o On the basis of confirmation

- Globular
- Fibrous

Action site

Modulating site (alloitric site)

Lock and key model Emil Fischer

Induced fit

Stained induced fit.

Isoenzymes

Factors affecting E catalyzed reactions:

o Substrate conc.

15

o Temperature

Q10 Temp, coefficient for every 10C  rate

doubled
Optimal temp.

o pH

(i)

E denaturation

(ii)

Alterations in the charged state of the E and/or

o Equilibrium

E SH+

ESH

E H+

EujH at low pH.

At high pH SH+

Not changed by an enzyme

Conc. -

o Key is a dynamic state.

Enzyme conc.

( [E]

16

S + H+

ENZYMES

General nature - Biocatalysis

o Protein increase the speed of a reaction.
o Act on specific called substrate to form an ES complex which is the changed
to produce:

Do not change during the reaction

Do not change the equilibrium constant of a reaction.

Increase the rate at which the reaction approaches equilibrium

Sensitive to effect of pH, temp ionic composition  are denatured

and lose activity

Apo E
catalytically
inactive

Cofactor or prosthetic 
gp

HbE
active

Small organic or inorganice

needed for activity mol. of E
Prosthetic gp is tightly bound to the E

Active site

V. specific -

Diff. enzymes differ in this

- Absolute specificity
- Group
- Reaction
- Optical specificity

All is added at the name of the E

Classification: International Union of Biochemistry:

6 major classes

Each divided into several subclasses which are further subdivided.

A No. is assigned to each class, subclass and sub-subclass.

17

Some trivial names

Class I Oxidoreductase

Involved in oxidation and reduction

e.g. alcohol NAD oxidoreductase catalysis conversion of alcohol to aldehyde bt

removing 2 e and 2 H.
H

Ho

R - C - OH + NAD+

'

R - C -H

+ NADH + H+

H
Dehydrogenase also act on the following functional groups as electron
donors:
- CH2 CH2 -

-CH2

-CH2 -CH = NH + Nucliotide

NADH + NADPH
Oxidase transfer 2 electron from the donor to ozygen resulting in H2H2
formation:
- D glucose  O2 

gluconolactone + H2O2

Ozygenase catalyse the incorporation of both atoms of O2 into a single

S. Hydrxtlases incorporate on atom of mol. O2 into the and the
second O2 appears as water.
Peroxidase use H2O2 as oxidant
NADH +

H+

H2O2 + H2O2 '

H2O2 as H+ donor and accepts

O2

2H2O

Class 2 Transferases

Transfer functional grps between donors and acceptors

18

Groups transferred include amino, acyl, phosphate one carbon and glycosynl
grps.
e.g. Transaminase : transfer = NH2 gp from amino acids to ketoacids to for a
new amino acid.

Kinases are phosphosylating Es that catalyse the transfer of the phsophoryl gp

from ATP to another nucleotide triphosphate, to alcohol or amino gp acceptor.
Glucokinase
ATP + Glucose  ADP

glucose-6-phosphate

Glycosyltransferase required for glycogen syn. catalyse the transfer of a

activated glycosyl residue to a glycogen primer.
UDP glucose + Glycogen  UDP + Glycogen
Primer (n)
(n+1)
Class 3 Hydrolases

Clear bonds by addition of water.

The general reactions involve the cleavage of C-O, C-N, O-P and C-S grps
O
R C NH R2

H2O

O
R C OH -+ NH2 R

Peptidase
Class 4 Lyases
Lyases remove or add water, ammonia or CO2 to produce bonds (in same cases)
Decarboxylases remove CO2
RCCO

R C H + CO2

19

Dehydrases remove H2O

CH2 Coo
H C Coo

Citrate dehydrase

CH2 Coo

CH2 Coo

C Coo + H2O

Citrate

HC Coo
Cis Aconitate

Class 5 Isomerases
Catalyse isomerization include cis-trans, lato-emol and aldose-ketose intercoversions.
Isomerases that catalyze the interconversion at asymmetric carbons are either
epimerases or racemerases.
CH2 OH
C=O

CH2 OH
Epimerases

HOCH

C=O
HCOH

CHOH

HCOH

H2CO PO3H2

H2COPO3H2

D-xylulose

S-Ribulose

5-Phosphate

5 Phosphate

COOH

Racemase

H COH

COOH
HOCH

CH2

CH3

D-Lactic Acid

L-Lactic Acid

Mutases catalyse the intramolecular transfer of a gp such as the phosphoxyl gp e.g.

phsophoglycerate mutase catalyzes the conversion of 2-phosphoglycerate to 3phosphoglycerate.

20

PGM
COOH

COOH

H C O (P)

H C OH

CH2OH

CH2O (P)

2-phosphoglycocerate

3-phosphoglycerate

Class 6 Ligases
Ligate means to bind. These E are involved in synthesis reactions where two
mol are joined at the expense of an ATP high energy phosphate bond. Also known as
synthetases
e.g. amino acid ERNA synthetases
Glutamine synthetase
Pyruvate carboxylase
COOH

COOH
(Bisline)

ATP + CO2

C=O

C = O + ADP + P

CH3

CH2
CooH

Pyr

Oxaloacetate

21

ENZYMES

Enzymes bind S at the active site to form on ES complex.

E are highly selective for S.

Active site is that region of a E that bind the S and the prosthetic group if any
contributes the residues that participate directly in making a breaking if bonds.
These residues are called the catalytic groups.

The active site is a relatively small part of the total vol. of an E.

The active site is 3D entity. It is not a point or online and is made of groups which
are far away from each other in the polypeptide chain.

S binds to the active site by weak forces.

Active sites are clefts or crevices from those water is usually excluded.

The specificity of binding depends on the precisely defined arrangement of atoms in

an active site.

Lock and key model

Active site has a shape complimentary to the active site.

Induced fit model. The active site has a shape complementary to that of the S only
after the S is bound.

22

Enzyme decrease the activation energey of reaction catalyzed by the

S'P

U G# = G transition G Substrate
state

Transition state has higher energy

the S or P
U G# Gibbs free energy of activation
E u the rate of a reaction by v U G#
Factors affecting the rate of E catalyzed reactions

Substrate
K1 K3
E + S ' ES t E + P
K2
V is and to S when S is very small
Km (Michaelis
constant)
V = K3 (ES)

K2 + K3
K1

(none of the ES goes back to E + S). The initial stage

of the reaction).

Rate of function of ES = K1 [E][S]

Rate of breakdown of ES = (K2 + K1) [ES]
L = steady state . Rate of formation of ES remains constant while S and P change.
K1 [E][S]
ES

K2 + K3) [ES]

[E][S]
(K2 + K3)/ K1
23

FACTORS AFFECTING RATE OF ENZYME CATALYZED REACTIONS

Enzyme Concentration
r [E]

Substrate concentration
Rate of catalysis K1, varies with [S].

Hypebolic way
When [S] is small, V is almost proportional to [S]. At high [S] is nearly
independent to [S]:

K1
E + S ' ES
K2
V=

K3


Proposed by Mchalis and Menten in 1913.

ES formed
None of the perverts back to ES a
condition that hold in the initial stages
of a reaction when P is .

E+P

K3 [ES]

Rate of formation of ES = K1 [E][S]

Rate of breakdown of ES = (K+2 + K+3) [ES]
We are interested in the catalytic rates under steady state conditions. In a steady
state, the concentration of intermediate stay the same while the concentration of
starting maternal and products are changing:
of
Rate of formation [ES] = rate of breakdown - [ES]
K1 [E][S] = (K+2 + K+3) [ES]

Km

[ES]

K+2 + K3
K1

Rearrange

[E][S]
(K+2 + K+3) [ES]
=

Michaelis constant

24

[S] ([E] (ES)]

[ES] Km

[S] [ES]

[ES] Km
[S]

Divide by [S]
=

Divide by [ES]
Et - 1
[ES]

[ES] = [E][S]
Km

Km
[S]

Et = Km + 1 =
[ES] [S}

Km + [S]

The concentration of [S] is nearly equal to total S concentration, provided

concentration of [E} is much lower that [S]. The concentration of uncombined [E]
is equal to
ES

([E] [ES]) (SS)

Km
solving to ES

or

ES

[Et]

[S] / Km
1+ [S] / Km

or

ES

[Et]

[S]
[S] + Km

or

K3 [Et]

[S]
[S] + Km

At a very high S concentration

or
equalation

[S]
= 1 (saturation of
[S] + Km
(S/with [E])

Vmax =

K3 [Et]

Vmax [5]

Michaelis Menlin

[S] + Km
At low [S] when [S] is much lower than Km
V

[5] Vmax
Km

25

i.e. rate x [S]

At high [S] whn [S] is much greater the Km

V

Vmax i.e. rate is maximal, independent of S soon.

when [S]

V=

Vmax
Km is equal to the
2
constant come at which the reaction rate is half the maximal value.
Vmax and Km can be determined by varying [S].
Reciprocal of Michaeles Menlin e.g.
1
V

1
Vmax

Km
Vmax

1
[S]

Significance of Km and Vmax

The Km form C depends on the particular [S] and on the environmental conditions e.g.
temp and ionic strength.
-

Km is the [S] concentration at Vmax at which half the active sites are filled.
Km: is the dissociation constant (consider K2 is much greater than K3 i.e.
dissociation of ES to E+P in which less than to [E] + [S]

K2 >> K3

Km is equal to the dissociation concentration of the ES complex if K3 is much

smaller than K2.

Km is the measure of the strength of ES. A high Km indicate weak binding, a low
Km indicates strong binding.

26

o Vmax reveals the turnover No. of E if the concentration of active site [Et] is
known
Vmax =

K3 [Et]

o The turnover No. of a enzyme is the No. of substrate mol. converted to

predict per unit time when the E is fully saturated with [S].
e.g. 10-6M sol. of carbonic anhydrase catalyses the formation of 0.6M
H2CO3 / sec when it is fully saturated with [S]
K3

6 x 105 sec -1

K3 is the turnover No.

The turnover No. 600,000 sec-1 for carbonic anhydrase is the
longest known.

Kinectus is the study of the rate of change of the initial state of reactant and products to
the final state of reactant and products (the term velocity, is often used).
Velocity is expressed in terms of change in the concentration of S or P / min
time.

27

No.

Class

Type of reaction catalyzed

1.

Oxidoreductase

Transfer of electron (Hydridian or H -----

2.

Transferase

Group transfer reaction

3.

Hydrolases

Hydrolyse in relation (transfer of functional

group to water).

4.

Lyases

Addition of groups to double and or additional

of double bonds by removal of groups.

5.

Isomerase

Transfer of groups with mol. to infield isomeric

forms

6.

Ligases

Formation of C-C, C-S, C-O and C-N bonds by

condensation reaction coupled to ATP
cleavage.

Oxidoreductase

A- + B

'

A + B-

Transferase

AB+C

'

A+B-C

Hydrolases

A B + H2O '

Lyases

Isomerase

Ligases

A+B

28

'

A H + B OH

AB

Y-X
'

AB

'

A-B

XY

INTERNATIONAL CLASSIFICATION OF ENZYMES

Suffix ase to the name of the S or the word or phrase describing their activity
e.g. increase acts on urea
protease
lipase
DNA polymerase
(some enzymes e.g. trypsin, pepsin, chymotrypsin do not denote their
substrates)

Some time some enzyme has 2 or more names and or 2 different enzymes have the
same names.

To avoid such a problem, there is an evere increasing number of enzymes, a system

for classification and naming of enzymes has been devised and adopted by
International Union of Biochemistry (IUB). According to this:
(i)

all enzymes are placed in 6 major chains, each with subclasses and subsubclasses, based on the type of reaction catalyzed (4-13 subclasses).

(ii)

The enzyme name has two parts. The first name the substrate or
substrates. the second, ending in ase, indicates the type of reaction
catalyzed.

(iii)

Additional information, if needed to clarify, is e.g. the co-enzyme

category) e.g. L. molate + NAD+ ' pyruvate + Cis + NADH + H is
designated 1.1.1.37 tomolate NAD+ xodiase reductase
(decarboxylating).

(iv)

Each E has a code No. (EC) that characterize the reaction type as to
which class (first digit), subclass (second digit) and sub-subclass (third
digit). the 4th digit for the specific E.
29

e.g. EC 2.7.1.1. denotes

Calss 2

Transferase

Subclass 7

Transfer of phosphate

Sub-subclass 1. An alcohol or phosphate acceptr.

Final digit

Hezokinase, or ATP: O-Hexose-6- phosphotransferase

30