Rafi M D: Textbook of Biochemistry for Medical Students (2 nd Edn) Universities Press

Enzyme Chemistry

Dr. Prakash
Associate Professor

Rafi M D: Textbook of Biochemistry for Medical Students (2 nd Edn) Universities Press

 Specific learning objectives
i. Active Center of Enzyme

ii. Factors affecting Enzyme activity

1) Enzyme concentration
iii. Enzyme Specificity
2) Substrate concentration

3) Product concentration
4) Effect of temperature
5) Effect of pH

6) Effect of activators
7) Effect of time
8) Effect of radiation
Rafi M D: Textbook of Biochemistry for Medical Students (2 nd Edn) Universities Press
 ACTIVE SITE
Active site (cleft-like portion )of an enzyme is the region that
binds substrates, co-factors and prosthetic groups and
contains residue that helps to hold the substrate and catalysis
occurs .
• Substrate binding site

• Catalytic site

A diagrammatic representation of
an enzyme and its active site

Rafi M D: Textbook of Biochemistry for Medical Students (2 nd Edn) Universities Press

 Active Site

Binding Site Catalytic Site

It chooses the substrate It performs the catalytic

and binds it to active site . action of enzyme.

Rafi M D: Textbook of Biochemistry for Medical Students (2 nd Edn) Universities Press

Salient features of active site
1. Existence of active site is due to the tertiary structure (three-
dimensional native conformation ) of protein.
2. Active site is made up of a. a. (catalytic residues) which are
far from each other in the linear sequence of a. a. (primary
structures of proteins). For instance, the enzyme lysozyme has
129 a. a. The active site is formed by the contribution of a. a.
residue numbered 35, 52, 62, 63, and 101.

Rafi M D: Textbook of Biochemistry for Medical Students (2 nd Edn) Universities Press

Salient features of active site

3. Active sites are regarded as clefts or cervices or pockets

occupying a small region (less than 5%) in a big enzyme.
4. Active site is not rigid in structure and shape. It is rather
flexible to promote the specific substrate binding and
function of enzyme.
5. Active site possesses a substrate binding site & a catalytic
site. The letter for the catalysis of the specific reaction.

6. The coenzymes or cofactors on Substrates binds at active site by

weak noncovalent bonds.

Rafi M D: Textbook of Biochemistry for Medical Students (2 nd Edn) Universities Press
Salient features of active site

7. Enzymes are specific in their function due to the existence of

active sites.

8. Commonly found a. a. at the active sites are serine, histidine,

cysteine, lysine, arginine, glutamate, tyrosine etc. Among
these a. a., serine is the frequently found.

9. E + S ↔ ES → P

Rafi M D: Textbook of Biochemistry for Medical Students (2 nd Edn) Universities Press

 Enzyme

s
E
Active
site E s

P
Rafi M D: Textbook of Biochemistry for Medical Students (2 nd Edn) Universities Press
 Enzyme Kinetic
 Velocity or rate of enzyme reaction :
 Rate of change of substrate to product per unit time
 The velocity is proportional to the concentration of
reacting molecules.
A+BC+D

 The maximum velocity (Vmax)

Maximum reaction rate attainable in presence of excess
substrate (at substrate saturation level)

 Km value
is substrate concentration (expressed in moles/L) at half-
maximal velocity

Rafi M D: Textbook of Biochemistry for Medical Students (2 nd Edn) Universities Press

 Enzyme Kinetic
Rectangular hyperbola
V max

V max /2

Km
S
The hyperbolic curve of reaction velocity (V) against substrate
concentration (S).
Km
Rafi M D: Textbook is the substrate
of Biochemistry concentration
for Medical Students
nd
(2 Edn) at ½ Vmax. Universities Press
 Factors affecting Enzyme activity

1) Enzyme concentration

2) Substrate concentration

3) Product concentration
4) Effect of temperature

5) Effect of pH
6) Effect of activators

7) Effect of time

8) Effect of radiation

Rafi M D: Textbook of Biochemistry for Medical Students (2 nd Edn) Universities Press

 Factors affecting Enzyme activity
1. Enzyme concentration
 Velocity (V) is directly proportional to the enzyme
concentration (sufficient substrate must be present)

E
Rafi M D: Textbook of Biochemistry for Medical Students (2 nd Edn) Universities Press
 DETERMINATION OF SERUM ENZYME

 The serum enzymes are determined for the

diagnosis of diseases.

By using a known volume of serum, and

keeping all the other factors (substrate, pH,
temperature etc.) at the optimum level, the
enzyme could be assayed in the laboratory

Rafi M D: Textbook of Biochemistry for Medical Students (2 nd Edn) Universities Press

 Factors affecting Enzyme activity
2. Substrate concentration
 Velocity (V) is directly proportional to the substrate
concentration (sufficient enzyme must be present)
3
After all enzyme
2 molecules are used up

3 phase reaction-
1) Linear
2) Curve
1 3) Plateau

V
S
Rafi M D: Textbook of Biochemistry for Medical Students (2 nd Edn) Universities Press
 Factors affecting Enzyme activity
• Substrate concentration
– The maximum velocity obtained is called Vmax.
– It represents the maximum reaction rate attainable in
presence of excess substrate.

Rafi M D: Textbook of Biochemistry for Medical Students (2 nd Edn) Universities Press

 Factors affecting Enzyme activity
3. Product concentration
 Velocity (V) is inversely proportional to the Product
concentration (Feedback mechanism)

Slowed, stopped or
reversed

V
p
Rafi M D: Textbook of Biochemistry for Medical Students (2 nd Edn) Universities Press
 3. Product concentration

a) The products combine with the active site of

enzyme and form a loose complex and, inhibit the
enzyme activity.

How to overcome product inhibition?

b) This type of inhibition is generally prevented by a

quick removal of products formed.

Rafi M D: Textbook of Biochemistry for Medical Students (2 nd Edn) Universities Press

 Factors affecting Enzyme activity
4. Effect of temprature
 Velocity (V) is maximum at Optimum temperature
 Increase or decrease in Temperature decreases the
activity of enzyme
r ve
Optimum
d cu
ape
ll sh
Be

V
Temperature

Rafi M D: Textbook of Biochemistry for Medical Students (2 nd Edn) Universities Press

 Factors affecting Enzyme activity
Effect of Temperature
 As temperature is increased towards optimum, the
reaction velocity is enhanced because:
 Activation energy (rate of motion) of molecules is
increased
 Interactions and collisions between substrate and
enzyme are increased

Optimum temperature- human enzymes have the

optimum temperature around 37°C(35-40oC)

Rafi M D: Textbook of Biochemistry for Medical Students (2 nd Edn) Universities Press

 Effect of Temperature
• Few enzymes (e.g. Taq DNA polymerase, muscle
adenylate kinase) are active even at 100°C.

What Happens When The Temp Is Raised

Beyond Optimum
 increase in temperature beyond the optimum
temperature causes a fall in the reaction velocity.
 This is because high temperature causes,
irreversible denaturation of the enzyme.

Rafi M D: Textbook of Biochemistry for Medical Students (2 nd Edn) Universities Press

 Temperature coefficient or Q10- is defined as
increase in enzyme velocity when the
temperature is increased by 10°C.
Majority of enzymes, Q10 = 2
between 0°C and 40°C.

Rafi M D: Textbook of Biochemistry for Medical Students (2 nd Edn) Universities Press

 Clinical significance –
A. Foods can be preserved in refrigerators (at
low temperatures) due to reduced bacterial
enzyme activities.
B. Certain surgeries are carried out by lowering
the patient’s body temperature (induced
hyporthermia), and thus the metabolic rate.

Rafi M D: Textbook of Biochemistry for Medical Students (2 nd Edn) Universities Press

 Factors affecting Enzyme activity
5. Effect of pH
 Velocity (V) is maximum at Optimum pH
 Increase or decrease in pH decreases the activity of
enzyme
rve
Optimum d cu
ape
l s h
l
Be

V
pH

Rafi M D: Textbook of Biochemistry for Medical Students (2 nd Edn) Universities Press

 Factors affecting Enzyme activity
Effect of pH on enzyme velocity
• Hydrogen ions influence the enzyme activity
by altering the ionic charges on the amino acids
(particularly at the active site), substrate, ES complex etc

OPTIMUM PH -may vary depending on the temperature,

concentration of substrate, presence of ions, etc.

-Most of the enzymes of higher organisms show optimum

activity around neutral pH (6-8).

Rafi M D: Textbook of Biochemistry for Medical Students (2 nd Edn) Universities Press

 Effect of pH

Rafi M D: Textbook of Biochemistry for Medical Students (2 nd Edn) Universities Press

 Factors affecting Enzyme activity
6. Effect of activators on Enzyme
• Some of the enzymes require certain inorganic metallic
cations like Mg2+, Mn2+, Zn2+, Ca2+, Co2+, Cu2+, Na+, K+
etc.
Metals function as activators of enzyme velocity through
various mechanisms—
A. Combining with the substrate,
B. Formation of ES-metal complex,
C. Direct participation in the reaction and bringing a
conformational change in the enzyme.

Rafi M D: Textbook of Biochemistry for Medical Students (2 nd Edn) Universities Press

 Two categories of enzymes requiring metals for
their activity are distinguished
• Metal-activated enzymes : The metal is not
tightly held by the enzyme and can be
exchanged easily with other ions
e.g. ATPase (Mg2+ and Ca2+),
Enolase (Mg2+)

Rafi M D: Textbook of Biochemistry for Medical Students (2 nd Edn) Universities Press

 2) Metalloenzymes : These enzymes hold the metals
tightly which are not readily exchanged.
e.g.
• Phenol oxidase (copper);
• Pyruvate oxidase (manganese);
• Xanthine oxidase (molybdenum);
• Cytochrome oxidase (iron and copper).

Rafi M D: Textbook of Biochemistry for Medical Students (2 nd Edn) Universities Press

 7. Effect of time

• Under ideal and optimal conditions (like pH,

temperature etc.), the time required for an
enzyme reaction is less.
Variations in the time of the reaction are
generally related to the alterations in pH and
temperature.

Rafi M D: Textbook of Biochemistry for Medical Students (2 nd Edn) Universities Press

 8. Effect of light and radiation
Exposure of enzymes to ultraviolet, beta,
gamma and X-rays inactivates certain enzymes
due to the formation of peroxides.
eg: UV rays inhibit salivary amylase activity.

Rafi M D: Textbook of Biochemistry for Medical Students (2 nd Edn) Universities Press

 Enzyme Specificity
 Thousands of enzymes present in the body
 They are highly specific in their action
 Specificity: Ability of an enzyme to discriminate between
two competing substrates
 Enzymes are highly specific for both
 Reaction catalyzed (Phosphorylation)
 Choice of substrates (Glucose)
The active site in a specified conformation on an intact enzyme
molecule is responsible

Rafi M D: Textbook of Biochemistry for Medical Students (2 nd Edn) Universities Press

 Types of Specificity
A. Stereo specificity:
 Stereospecific- acts only on one isomer

 Act on Particular linkage and particular stereo isomer

 e.g.

a. L-amino acid oxidase acts on L-amino acids

D-amino acid oxidase acts on D-amino acids
b. Lactate dehydrogenase, acting on pyruvate will form
only L-lactate, but not the D-lactate.

Rafi M D: Textbook of Biochemistry for Medical Students (2 nd Edn) Universities Press

Types of Specificity
B. Substrate specificity:
 Absolute substrate specificity:
 Acts only on 1 substrate
 e.g. Glucokinase: phosphorylates glucose only,
Urease splits urea to ammonia and carbon dioxide.
 Relative substrate specificity:
 Certain enzymes act on >1 substrate which are
structurally related.
 e.g. Hexokinase phosphorylates glucose, fructose
and mannose.
Rafi M D: Textbook of Biochemistry for Medical Students (2 nd Edn) Universities Press
 Types of Specificity
C. Bond specificity:
 Act on particular bond (One group)

 Proteolytic Enzyme: Peptide Bond

 Glycosidases: Glycosidic bonds

 Lipases: Ester bonds

D. Dual specificity:

1. One Enzyme - Two substrate :

2. One enzyme-Two reactions:

Rafi M D: Textbook of Biochemistry for Medical Students (2 nd Edn) Universities Press

 Types of Specificity
E. Reaction specificity:
 The same substrate can undergo different types of
reactions, each catalyzed by a separate enzyme.

Acetyl CoA
DH
P
LDH
Pyruvate PC Lactate

Oxaloacetat
e
Rafi M D: Textbook of Biochemistry for Medical Students (2 nd Edn) Universities Press
Rafi M D: Textbook of Biochemistry for Medical Students (2 nd Edn) Universities Press