CLASSIFICATION OF ENZYMES 5m

Enzymes are biocatalysts that are essential for biochemical recations to procedd in the human body

Biological activity of enzymes is dependent on the structural conformation of the protein

Almost all enzymes are proteins

Enzymes follow all chemical and physical properties of proteins

They are heat labile

They are water solouble

International Union of Biochemistry and Molecular Biology (IUBMB suggested the IUBMB system of nomenclature of
enzymes. It is complex and cumbersome; but unambiguous. As per this system, the name starts with EC (enzyme class)
followed by 4 digits.

First digit represents the class

Second digit stands for the subclass
Third digit is the sub-sub class or subgroup
Fourth digit gives the number of the particular enzyme in
the list. Enzymes can be classified into six types

Oxidoreductases: This class of enzymes will catalyze oxidation of one

substrate with simultaneous reduction of another substrate
or co-enzyme. This may be represented as
AH2 + B A + BH2
for example,
Alcohol + NAD+ Aldehyde + NADH + H+
1. The enzyme is Alcohol dehydrogenase

Other examples of oxidoreductases are succinate dehydrogenase and lacate dehydrogenase

Trasnferases: This class of enzymes transfers one group (other than

hydrogen) from the substrate to another substrate. This
may be represented as
A-R + B → A + B-R , For example,
Hexose + ATP → Hexose-6-phosphate + ADP

The enzyme is hexokinase

Hydrolases: This class of enzymes can hydrolyze ester, ether, peptide

or glycosidic bonds by adding water and then breaking the
bond.
Acetylcholine + H2O → Choline + acetate
The enzyme is Acetylcholine esterase
. All digestive enzymes are
hydrolases.

Lyases: These enzymes can remove groups from substrates or break

bonds by mechanisms other than hydrolysis. For example,
Fructose-1,6-bisphosphate → Glyceraldehyde-3-phosphate
+dihydroxy acetone phosphate
The enzyme is Aldolase

Isomerases: These enzymes can produce optical, geometric or positional

isomers of substrates. Racemases, epimerases, cistrans
isomerases are examples.
Glyceraldehyde-3-phosphate → Dihydroxy acetone
phosphate
Enzyme is Triose phosphate isomerase

Ligases: These enzymes link two substrates together, usually with

the simultaneous hydrolysis of ATP, For example,
Acetyl CoA + CO2 + ATP → Malonyl CoA + ADP +Pi
Enzyme is Acetyl CoA carboxylase.

Km value 5m

According to michelis theory theFORMATION of enzyme substrate complex is reversible but the breakdown of enzyme
substrate complex is irreversible

Km is independent of enzyme concentration

If enzyme concentration is doubled the vmax will double. but he km will remain exactly the same
Km value is substrate concentration (expressed in moles/L) at half-maximal velocity
2. It denotes that 50% of enzyme molecules are bound with
substrate molecules at that particular substrate concentration
4. Km is the signature of the enzyme. Km value is thus a
constant for an enzyme. It is the characteristic feature of a
particular enzyme for a specific substrate.
5. The affinity of an enzyme towards its substrate is inversely
related to the dissociation constant, Kd for the enzyme substrate
complex.
6. Km denotes the affinity of enzyme for substrate. The
lesser the numerical value of Km, the affinity of the enzyme for
the substrate is more.
Box 5.8: Salient features of Km
Fig. 5.14:

Effect of enzyme concentration on km effect of substrate concentration (substrate saturation curve)

FACTORS AFFECTING ENZYME ACTIVITY

1. Enzyme concentration: Rate of a reaction or velocity (V) is directly

proportional to the enzyme concentration, when
sufficient substrate is present
Hence, this property is made use of determining the
level of particular enzyme in plasma, serum or tissues.
2. Substrate concentration: As substrate concentration is increased, the velocity is
also correspondingly increased in the initial phases; but
the curve flattens afterwards
At lower concentrations of substrate (point A in the curve), some enzyme molecules are remaining idle. As
substrate is increased, more and more enzyme molecules are working. At half-maximal velocity, 50% enzymes
are attached with substrate (point B in the curve). As more substrate is added, all enzyme molecules are
saturated (point C). Further increase in substrate cannot make any effect in the reaction velocity (point D). The
maximum velocity obtained is called Vmax (Fig. 5.13B). It represents the maximum reaction rate attainable in
presence of excess substrate (at substrate saturation level).

3. Product concentration: In a reversible reaction, S P, when equilibrium is reached,

as per the law of mass action, the reaction rate is slowed
down. So when product concentration is increased, the reaction
is slowed, stopped or even reversed. In inborn errors of
metabolism, one enzyme of a metabolic pathway is blocked
.
For example,
A _E_1_ B _E_2_ C —E2_ __ D
If E3 enzyme is absent, C will accumulate, which in
turn, will inhibit E2. Consequently, in course of time, the
whole pathway is blocked.
4. Temperature: The velocity of enzyme reaction increases when temperature
of the medium is increased; reaches a maximum and then
falls (Bell shaped curve).
The temperature at which
maximum amount of the substrate is converted to the
product per unit time is called the optimum temperature
(Fig. 5.17). As temperature is increased, more molecules
get activation energy, so the reaction velocity is enhanced.
The temperature
coefficient (Q10) is the factor by which the rate of catalysis
is increased by a rise in 10°C. Generally,
the rate of reaction of most enzymes will
double by a rise in 10°C.
But when temperature is more than 50°C, heat denaturation and consequent
loss of tertiary structure of protein occurs.
. Most human enzymes have the optimum temperature around 37°C.
Certain bacteria living in hot springs willhave enzymes with optimum temperaturenear 100°C.

5. Hydrogen ion concentration (pH): Each enzyme has an optimum pH, on both sides of which
the velocity will be drastically reduced. The graph will
show a bell shaped curve (Fig.5.18). The pH decides the
charge on the amino acid residues at the active site. The
net charge on the enzyme protein would influence substrate
binding and catalytic activity. Optimum pH may vary
depending on the temperature, concentration of substrate,
presence of ions, etc. Usually enzymes have the optimum pH between 6 and 8. Some important exceptions are pepsin
(with optimum pH 1–2); alkaline phosphatase (optimum
pH 9–10) and acid phosphatase (4–5).

6. Presence of activators: Some of the enzymes require certain

inorganic metallic cations like Mg2+, Mn2+,
Zn2+, Ca2+, Co2+, Cu2+, Na+, K+ etc. for their
optimum activity. Rarely, anions are also needed
for enzyme activity e.g. chloride ion (Cl–)
for amylase. Metals function as activators of
enzyme velocity through various mechanisms
and bringing a conformational change
in the enzyme.
Two categories of enzymes requiring metals
for their activity are distinguished
l 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+)
l Metalloenzymes : These enzymes hold

the metals rather tightly which are not

readily exchanged. e.g. alcohol dehydrogenase,
carbonic anhydrase, alkaline phosphatase,
carboxypeptidase and aldolase
contain zinc.
Cytochrome oxidase (iron and copper).
7. Effect
7. Presence of inhibitors: Enzyme inhibitor is defined as a substance
which binds with the enzyme and brings about
a decrease in catalytic activity of that enzyme.
The inhibitor may be organic or inorganic in
nature. There are three broad categories
enzyme inhibition
1. Reversible inhibition.
Competitive uncompetitive non competitive
2. Irreversible inhibition.[suicide inhibition]
3. Allosteric inhibition.
8. Presence of repressor or de repressor:
Even though both inhibition and repression reduce the
enzyme velocity, the mechanisms are different In the case
of inhibition, the inhibitor acts on the enzyme directly;on the contrary repressor acts at the gene level;
the effect is noticeable only after a lag period of hours or
days; and the number of enzyme molecules is reduced in
the presence of repressor molecule.

9. Covalent modification: The activity of enzymes may be increased or decreased by

covalent modification. It means, either addition of a group
to the enzyme protein by a covalent bond; or removal of a
group by cleaving a covalent bond.
Zymogen activation by partial proteolysis is an
example of covalent activation.
This is a reversible reaction.
The most common type of covalent modification is the
reversible protein phosphorylation.
.

Enzyme inhibition type description and examples

Coenzymes
The non-protein, organic, low molecular
weight and dialysable substance associated with
enzyme function is known as coenzyme.
It is heat stable. non-protein part, called the
prosthetic group.
ii. The protein part of the enzyme is then named the apoenzyme.
It is heat labile.
iii. These two portions combined together are called the
holo-enzyme.
v. Co-enzymes may be divided into two groups
a. Those taking part in reactions catalyzed by
oxidoreductases by donating or accepting hydrogen
atoms or electrons. Therefore, such
co-enzymes may be considered as co-substrates or
secondary substrates
NADP–NADPH; FAD-FADH2
and FMN–FMNH2.
b. Those co-enzymes taking part in reactions transfer
ring groups other than hydrogen
Most
of them belong to vitamin B complex group
Thiamine pyrophosphate (TPP)
Pyridoxal phosphate (PLP)
Biotin
Carbon dioxide
Co-enzyme-A (Co-A)
Adenosine triphosphate (ATP)
1. The protein part of the enzyme gives the necessary three dimensional
infrastructure for chemical reaction; but the
group is transferred from or accepted by the co-enzyme
2. The co-enzyme is essential for the biological activity of the
enzyme
5. Inside the body, when the reaction is completed, the coenzyme
is released from the apo-enzyme, and can bind to
another enzyme molecule the reduced co-enzyme, generated in the first reaction can
take part in the second reaction. The coupling of these two
reactions becomes essential in anaerobic glycolysis
for regeneration of NAD+
6. One molecule of the co-enzyme is able to convert a large
number of substrate molecules with the help of enzyme
Isoenzymes
Iso-enzymes of LDH, CK, ALP; enzymes used for therapeutic and
diagnostic purposes
The multiple forms of an enzyme catalysing
the same reaction are isoenzymes or isozymes.
They, however, differ in their physical and
chemical properties which include the structure,
electrophoretic and immunological properties,
Km and Vmax values, pH optimum, relative
susceptibility to inhibitors and degree of
denaturation.
Isoenzymes of alkaline phosphatase
As many as six isoenzymes of alkaline
phosphatase (ALP) have been identified. ALP is
a monomer, the isoenzymes are due to
the difference in the carbohydrate content
(sialic acid residues). The most important
ALP isoenzymes are 􀁄1-ALP, 􀁄2-heat labile
ALP, 􀁄2-heat stable ALP, pre-􀁅 ALP, 􀁊-ALP
etc.
Increase in 􀁄2-heat labile ALP suggests
hepatitis whereas pre 􀁅-ALP indicates bone
diseases.
Isoenzymes of creatine
phosphokinase
Creatine kinase (CK) or creatine phosphokinase
(CPK) catalyses the inter-conversion of phosphocreatine
(or creatine phosphate) to creatine.
Creatine
CPK
ADP ATP
Phosphocreatine
CPK exists as three isoenzymes. Each
isoenzyme is a dimer composed of two
subunits—M (muscle) or B (brain) or both.
Isoenzyme Subunit Tissue of origin
CPK1 BB Brain
CPK2 MB Heart
CPK3 MM Skeletal muscle
In healthy individuals, the isoenzyme
CPK2 (MB) is almost undetectable in serum
with less than 2% of total CPK. After the
myocardial infarction (MI), within the first 6-18
hours, CPK2 increases in the serum to as high as
20% (against 2% normal). CPK2 isoenzyme is
not elevated in skeletal muscle disorders.
Therefore, estimation of the enzyme CPK2 (MB)
is the earliest reliable indication of myocardial
infarction.
Isoenzymes of lactate
dehydrogenase (LDH)
LDH whose systematic name is L-lactate-
NAD+ oxidoreductase (E.C. 1.1.1.27) catalyses
the interconversion of lactate and pyruvate as
shown below

LDH has five distinct isoenzymes LDH1,

LDH2, LDH3, LDH4 and LDH5.
LDH is an
oligomeric (tetrameric) enzyme made up of four
polypeptide subunits. Two types of subunits
namely M (for muscle) and H (for heart) are
produced by different genes. M–subunit is basic
while H subunit is acidic.

Diagnostic importance of LDH : Isoenzymes

of LDH have immense value in the diagnosis of
heart and liver related disorders (Fig.6.15). In
healthy individuals, the activity of LDH2 is
higher than that of LDH1 in serum. In the case of
myocardial infarction, LDH1 is much greater
than LDH2 and this happens within 12 to 24
hours after infarction. Increased activity of LDH5 in serum is an indicator of liver diseases. LDH
activity in the RBC is 80–100 times more than
that in the serum. Hence for estimation of LDH
or its isoenzymes, serum should be totally free
from hemolysis or else false positive results will
be obtained.
Therapeutic enzymes& Diagnostic enzymes

Enzymes as Therapeutic Agents

Streptokinase (from Streptococcus) or Urokinase (from urine) lyse intravascular clots and are therefore used
can in myocardial infarction. Pepsin and trypsin are given to patients with defective digestion. Asparaginase is
used as an anticancer drug. A list of therapeutically useful enzymes is given in Table 4.12.

Enzymes Used for Diagnosis

A list of enzymes used in clinical laboratory is given in Table 4.13. Blood estimations are made specific by using
enzymes. For example, glucose oxidase is used to estimate glucose. Urease will act only on urea, and
therefore, it is conveniently used to quantitate urea in biological fluids. The presence of antibody in circulation
is identified by fixing them on antigen and identified by a second antibody tagged with peroxidase. These
enzymes are effective to produce a color reaction. This ELISA test is described in detail in Chapter 44.
Restriction endonucleases are used to cut DNA at specific sites; and applied in recombinant DNA technology,
Southern blotting, and other advanced molecular biology techniques