THEME:

Biochemistry - as science; biomolecules;

metabolic pathways. Structure and properties
of enzymes. The mechanism of enzymes
activity. I soenzymes. Classification of enzymes.
PLAN OF THE LECTURE
Naming and Classification of Enzymes
Enzyme Cofactors
Chemical Kinetics
The Free Energy of Activation
Kinetics of Enzyme-Catalyzed Reactions
The Michaelis-Menten Equation
Effect of pH on Enzymatic Activity
Effect of Temperature on Enzymatic Reactions
Enzyme Inhibition (Reversible)
Enzyme Inhibition (Irreversible)
Isoenzymes
Enzyme-Activity Units
Definition
Enzymes are protein catalysts for
biochemical reactions in living cells
They are among the most remarkable
biomolecules known because of their
extraordinary specificity and catalytic
power, which are far greater than those of
man-made catalysts.
Naming

").
The name enzyme (from Greek "in yeast")
was not used until 1877,
but much earlier it was suspected that
biological catalysts
are involved in the fermentation of sugar
to form alcohol
(hence the earlier name "ferments").
Naming and Classification of
Enzymes

Many enzymes have been named by adding the
suffix -ase to the name of the substrate, i.e., the
molecule on which the enzyme exerts catalytic
action.

For example, urease catalyzes hydrolysis of
urea to ammonia and CO
2
, arginase catalyzes
the hydrolysis of arginine to ornithine and
urea, and phosphatase the hydrolysis of
phosphate esters.
Classification of enzymes
Oxido-reductases (oxidation-reduction reaction).
Transferases (transfer of functional groups).
Hydrolases (hydrolysis reaction).
Lyases (addition to double bonds).
Isomerases (izomerization reactions).
Ligases (formation of bonds with ATP
cleavage).
The structure of enzymes
Protein part + Non- protein part
Apoenzyme + Cofactor = Holoenzyme

Function of apoenzyme:
It is responsible for the reaction
Function of cofactor:
It is responsible for the bonds formation between
enzyme and substrate
Transfer of functional groups
Takes plase in the formation of tertiary structure of
protein part
Cofactor
1. Prosthetic group (when cofactor is very
tightly bound to the apoenzyme and has
small size )
2. Metal ion
3. Coenzyme(organic molecule derived
from the B vitamin which participate
directly in enzymatic reactions)
Prosthetic group
1. Heme group of cytochromes

2. Biocytin group of acetyl-CoA
carboxylase
Metal ions
Fe - cytochrome oxidase, catalase
Cu - cytochrome oxidase, catalase
Zn - alcohol dehydrogenase
Mg - hexokinase, glucose-6-phosphatase
K, Mg - pyruvate kinase
Na, K ATP-ase

Coenzyme
B
1
TPP- Thiamine Pyro Phosphate
B
2

FAD- Flavin Adenine Dinucleotide
FMN- Flavin Mono Nucleotide
Pantothenic acid
Coenzyme A (CoA)
B
5
NAD Nicotinamide Adenine Dinucleotide
NADP- Nicotinamide Adenine Dinucleotide Phosphate


Chemical Kinetics

Chemical reactions may be classified on the basis of the
number of molecules that must ultimately react to form
the reaction products. Thus, we have monomolecular,
bimolecular, and termolecular reactions, in which one,
two, or three molecules, respectively, undergo reaction.
Chemical reactions are also classified on a kinetic basis,
by reaction order, and we have zero-order, first-order,
second-order, and third-order reactions, depending on
how the reaction rate is influenced by the concentration
of the reactants under a given set of conditions.
First-order reactions
are those which proceed at a rate exactly
proportional to the concentration of one
reactant. The simplest example is when the rate
of the reaction
A> P
is exactly proportional to the concentration of A.
Second-order reactions
are those in which the rate is proportional to
the product of the concentrations of two
reactants or to the second power of a single
reactant. The simplest example is the
reaction
A + B > P.
Third-order reactions
which are relatively rare, are those whose
velocity is proportional to the product of
three concentration terms.
A + B + C > P.

Zero-order reactions
Some chemical reactions are independent of
the concentration of any reactant; these are
called zero-order reactions.
The Michaelis-Menten
Equation
In 1913 a general theory of enzyme action and kinetics
was developed by Leonor Michaelis and Maud Menten.

The enzyme E firstly combines with the substrate S to
form the enzyme-substrate complex ES; the latter then
breaks down in a second step to form free enzyme and
the product P:
E+S ES (1)
ES E + P (2)
Mechanism of enzyme reaction
1. Formation of enzyme substrate
complex
E + S ES
2. Conversion of the substrate to the product
ES EP
3. Release of the product from the enzyme
EP E+P
Effect of substrate concentration
on the rate of an enzyme-
catalyzed reaction.

The Free Energy of
Activation
Before a chemical reaction can take place, the
reactants must become activated.
This needs a certain amount of energy which is
termed the energy of activation.
It is defined as the minimum amount of energy
which is required of a molecule to take part in
a reaction.
The Free Energy of
Activation
For example,decomposition of hydrogen
peroxide without a catalyst has an energy
activation about 18 000. When the enzyme
catalase is added, it is less than 2000.
The Free Energy of
Activation
The rate of the reaction is proportional to
the energy of activation:
Greater the energy of activation
Slower will be the reaction
While if the energy of activation is less,
The reaction will be faster
Energy of Activation

The Free Energy of
Activation
Reactants A and B, in order to react to form
the product C, must pass through a
transition state (A-B) in which the activated
complex is formed and certain amount of
activation energy(Ea) is required. When the
proper enzyme is present, less energy is
required for activation (Ea).
Effect of pH on Enzymatic
Activity

Most enzymes have a characteristic pH at
which their activity is maximal (pH-
optimum);
above or below this pH the activity
declines. Although the pH-activity profiles
of many enzymes are bell-shaped, they may
be very considerably in form.
Effect of pH on Enzymatic
Activity

Effect of Temperature on
Enzymatic Reactions

.The rate of enzyme catalysed reaction generally
increases with temperature range in which the
enzyme is stable. The rate of most enzymatic
reactions doubles for each 10
0
C rise in
temperature. This is true only up to about 50
0
C.
Above this temperature, we observe heat
inactivation of enzymes.
The optimum temperature of an enzyme is that
temperature at which the greatest amount of
substrate is changed in unit time.

Effect of Temperature on
Enzymatic Reactions
Enzyme Inhibition
1. Reversible inhibition
A. Competitive
B. Non-competitive
C. Uncompetitive

2. Irreversible inhibition
Competitive Inhibition
Competitive inhibition involves a compound
which resembles the substrate in structure and
binds to the catalytic site of the enzyme. The
substrate is then prevented from binding to the
catalytic site of the enzyme.
The formation of an enzyme-inhibitor complex
may be represented as
E + I = EI
Usage competitive inhibition in
medicine
The antibacterial effects of sulfanilamides
are also explained by their close
resemblance to para-amino-benzoic acid
which is a part of folic acid, an essential
normal constituent of bacterial cells. The
sulfanilamides inhibit the formation of folic
acid by bacterial cells and thus the bacterial
multiplication is prevented and they soon
die.
Non-competitive Inhibition
In this case, there is no structural
resemblance between the inhibitor and the
substrate. The inhibitor does not combine
with the enzyme at its active site but
combines at some other site.

E + S +I =ESI (INACTIVE COMPLEX)
Uncompetitive inhibition
The inhibitor does not combine with the
free enzyme but it combines only with the
enzyme-substrate complex forming inactive
enzyme-substrate-inhibitor complex.
E + S = ES
ES + I = ESI
Irreversible Inhibition
The inhibitor is covalently linked to the
enzyme.
The example:
Action of nerve gas poisons on
acetylcholinesterase,an enzyme that has an
important role in the transmission of nerve
impulse.
These are the enzymes from the same
organism which catalyse the same reaction
but are chemically and physically distinct
from each other.

Isoenzymes





Lactate dehydrogenase
It occurs in 5 possible forms in the blood
serum:
LDH
1
LDH
2
LDH
3
LDH
4
LDH
5
Structure of LDH
Each contains 4 polypeptide chains which
are of 2 types: A and B which are usually
called M (muscle) and H (heart).
LDH
1
H H H H

LDH
2
H H H M

LDH
3
H H M M

LDH
4
H M M M

LDH
5
M M M M



Clinical importance of LDH
Acute myocardial infarction
LDH
1
and LDH
2

Acute liver damage
LDH
4
and LDH
5
Creatine kinase
It has 3 isoenzymes:
CK
1
CK
2
CK
3

Clinical importance:
When patient have acute myocardial infarction
CK appears in the blood 4 to 8 hours after onset of
infarction and reaches a peak in activity after 24
hours.
Enzyme-Activity Units

The most widely used unit of enzyme activity is
international unit defined as that amount which
causes transformation of 1.0 mkmol of substrate
per minute at 25C under

The specific activity is the number of enzyme units
per milligram of protein.

Enzyme-Activity Units

The molar or molecular activity, is the
number of substrate molecules transformed
per minute by a single enzyme molecule

The katal (abbreviated kat), defined as the
amount of enzyme that transforms 1 mol of
substrate per 1 sec.