Enzymes for UNDERGRADUATES AS PER RECENT NMC CURICULLUMRRI

Definition
 Enzymes may be defined as protein biocatalysts
that catalyse chemical reactions in biological
systems, synthesized by living cells, colloidal and
thermolabile in character and specific in their
action
 But this definition is not entirely correct, as some
RNA molecules, called ribozymes, have now
been found to catalyse some biochemical
reactions

 The reactant on which the enzyme acts is known
as the substrate of the enzyme
 The enzyme converts the substrate into a
product
Substrate Product
Enzyme

 Berzelius in 1836 coined the term catalysis
(Greek: to dissolve)
 In 1878, Kuhne used the word enzyme
 isolation of enzyme system from cell-free extract
of yeast was achieved in 1883 by Buchne
 James Sumner (1926) first achieved the isolation
and crystallization of the enzyme urease from jack
bean and identified it as a protein

 Enzyme (conjugated protein part)=Apo-enzyme
(inactive)
 The non-protein part = Co-enzyme
 Apo-enzyme + Co-enzyme= Holoenzyme
(active)

Apo-Enzyme and Co-Enzyme
 Some enzymes have two parts
 The protein part is called Apoenzyme
 The non-protein part is called Co-enzyme
 When tightly bound the Co-enzyme is called
Prosthetic-Group

COENZYMES AND COFACTORS
 Some enzymes require the presence of a non-
protein substance for their catalytic activity
 The non-protein, organic, low molecular weight,
dialyzable substance associated with enzyme
function is known as a coenzyme
 If it is inorganic, it is known as a cofactor

 The coenzyme or the cofactor may be an integral
part of the enzyme molecule or its presence may
be required during the reaction
 The protein portion of an enzyme that requires a
coenzyme is called apoenzyme
 The apoenzyme combines with the coenzyme to
form the holoenzyme which is the catalytically
active form of the enzyme:
Apoenzyme + Coenzyme Holoenzyme

Role of Coenzymes
 The enzyme acts upon its substrate, and
converts it into a product
 The coenzyme may be regarded as a co-
substrate or a second substrate in the group
transfer reactions
 The coenzyme participates in the reaction either
as a donor or as an acceptor of the group that is
being transferred

Coenzymes of B- complex vitamin
Coenzymes
1. FMN
2. FAD
3. NAD+
4. NADP+
5. Lipoic acid
6. PLP
7. Coenzyme A
8. Tetrahydrofolate
9. TPP
10. Methylecobalamine
Atom or group transfer
1. H/ē
2. H/ē
3. H/ē
4. H/ē
5. H/ē
6. Amino/ keto
7. Acyl
8. Formyl, methenyl
9. Aldehyde/keto
10. Methyle
1. L-amino acid oxidase
2. D-amino acid oxidase
3. LDH
4. G-6-PO4 dehydrogenase
5. PDH, α-KGD
6. ALT, AST
7. Thiokinase
8. Formyl transferase
9. Transketolase
10. Methylmalonyl CoA
mutase
Dependant enzymes

Coenzymes non B- complex vitamin
Coenzymes
1. ATP
2. CDP
3. UDP
4. SAM
5. PAPS
(Phoshoadenosine
phosphosulfate)
1. Donate PO4--, adenosine, AMP
moieties
2. Phospholipids synthesis choline
and ethanolamine
3. Carrier of glucose, Galactose and
glycogen synthesis
4.Donate methyl group
5. Mucopolysaccharides synthesis
Biochemical function

Co-Enzyme Vitamin
Derivative
Enzyme Requiring
TPP
Co-Decarboxylase Vitamin B1
Thiamine
Pyruvate Dehydrogenase
α -Ketoglutarate
Dehydrogenase
Transketolase
FAD
FMN
Vitamin B2
(Riboflavin)
D-amino acid Oxidase
L-amino acid Oxidase
Pyridoxal PO4
Co-
Transaminase
Vitamin B6
Pyridoxine Transaminase

Co-Enzyme Vitamin
Derivative
Enzyme Requiring
Biotin
Co-Carboxylase
Vitamin
Biotin
Acetyl CoA Carboxylase
Pyruvate Carboxylase
Ubiquinone
NADH Dehydrogenase
Cytochrome b
Heme
Co-enzyme
- Peroxidase
Catalase
Cytochrome

ENZYME NOMENCLATURE AND CLASSIFICATION
 The nomenclature of enzymes has undergone
many changes over the years
 The names assigned to enzymes in the
beginning were very vague and uninformative
 Some of the earliest names, e.g. pepsin, ptylin,
zymase etc, indicate neither the substrates of the
enzymes nor the type of reactions catalysed by
them

 Later on, a slightly more informative
nomenclature was adopted
 Suffix -ase was added to the name of the
substrate e.g. lipase, protease, ribonuclease etc
 Still the type of reaction catalysed by the
enzyme remained unclear

 Nomenclature was modified further, to include
the name of the substrate followed by the type
of reaction ending with -ase
 This resulted in names like lactate
dehydrogenase, pyruvate carboxylase,
glutamate decarboxylase etc
 Even these names do not give complete
information about the reaction, for example
whether a coenzyme is required or a byproduct
is formed

 To make the names of enzymes precise, fully
informative and unambiguous, International
Union of Biochemistry (IUB) devised a
method of nomenclature and classification
of enzymes

According to IUB system:
1. The enzymes have been divided into six
classes (numbered 1-6)
2. Each class is divided into subclasses (1-9)
3. Subclasses are divided into subsubclasses
4. Subsubclasses are divided into individual enzymes

 The name of the enzyme has two parts
 The first part includes the name(s) of the
substrate(s) including coenzyme(s) if they
act as second substrate(s)
 The second part includes the type of reaction
ending with -ase
 If any additional information is to be given, it is
put in parenthesis at the end

 For example, the enzyme having the trivial
name glutamate dehydrogenase catalyses the
following reaction :
L-Glutamate + NAD(P) + H O
+
2
a-Ketoglutarate + NAD(P)H H + NH
+
3
+
 According to IUB system, this enzyme in
known as L-Glutamate: NAD (P)
oxidoreductase (deaminating)

 This name shows that the enzyme acts on L-
glutamate
 NAD+ or NADP+ is required as a co-substrate
 The type of reaction is oxidoreduction i.e. L-
glutamate is oxidised and the co-substrate is
reduced
 The amino group of L-glutamate is released as
ammonia

 Thus, the name gives complete information
about the reaction catalysed by the enzyme
 Moreover, each enzyme has been given a code
number consisting of four digits which,
successively, denote the number of the
class, subclass, subsubclass and the individual
enzyme

 The code number of L-glutamate: NAD(P)
oxidoreductase (deaminating) is EC 1.4.1.3
which shows that is it the third enzyme of
subsubclass 1 of subclass 4 of class 1
 EC is the acronym for Enzymes Commission

According to IUB classification, the enzymes
are divided into the following six classes:
1. Oxidoreductases
2. Transferases
3. Hydrolases
4. Lyases
5. Isomerases
6. Ligases
OTHLIL

Oxidoreductases
 These are the enzymes that catalyse oxidation-
reduction reactions
 One of the substrates is oxidised and the other is
reduced
 There are several subclasses of
oxidoreductases each acting on a particular
chemical group

 The groups undergoing oxidation-reduction
include – CH=CH –, CH – OH, C=O, CH – NH2 etc
 Examples of oxidoreductases are glutamate
dehydrogenase, lactate dehydrogenase,
malate dehydrogenase, glycerol-3-phosphate
dehydrogenase, cytochrom oxidase, L- and D
amino acid oxidases, alcoholic dehydrogenase
(alcohol: NAD+ oxidoreductase, E.C. 1.1.1.1.),

xanthine oxidase, glutathione reductase,
glucose-6-phosphate dehydrogenase etc

These can further be sub-classified as
(1.1) Those acting on CH-OH of donors
eg: Glycerol-3-phosphate Dehydrogenase
Glycerol-3-P + NAD Di Hydroxyacetone -P
+ NADH
Other eg:-- Lactate Dehydrogenase
Malate Dehydrogenase
Isocitrate Dehydrogenase
Alcoholic Dehydrogenase

(1.2)Those acting on CHO group of donors
eg. Glyceraldehyde-3-phosphate Dehydrogenase
(1.3)Those acting on CH-CH group of donors
eg: Dihydrouracil dehydrogenase
4,5 dihydrouracil + NAD+ Uracil + NADH
(1.4) Those acting on CH-NH2 group of donor
with NAD or NADP as acceptor.
eg: Glutamate Dehydrogenase
Glutamate + H2O + NAD+ 2 Oxoglutarate
NH + NADH

Transferases
 These enzymes transfer a group other than
hydrogen from one substrate to another
 Such groups include methyl group, amino group,
phosphate group, acyl group, glycosyl group
 Examples include hexokinase (ATP: D-hexose 6
phoshotransferse, E.C. 2.7.1.1.), glucokinase,
SGOT, SGPT, ornithine carbamoyl
transferase, transmethylase, Phosphoglucomutase,
hexose-1-phosphate uridyltransferase, CPK
(2.7.3.2.) etc

(2.1) Methyl Transfer: Methyl transferases
a. S-adenosyl Methionine + Guanidino acetate
Guanidino acetate Methyl transferase
S-adenosyl homocysteine + Creatine
b. Serine hydroxy methyl transferase
Serine Glycine

(2.2) Aldehyde, Keto Residue Transfer--eg: Transaldolase,
Transketolase
(2.3) Acyl Transferases—eg: Transacylases
(2.4) Glucosyl Transfer--eg: Transglucosylase
(2.6) Amino Grp Transfer--eg: Transaminases (SGOT, SGPT)
(2.7) Phosphorus Transfer eg: Glucokinase, Hexokinase

Hydrolases
 These are hydrolytic enzymes which hydrolyse
bonds such as peptide, ester, glycosidic bonds
etc
 These enzymes are commonly found in the
digestive secretions and lysosomes

 They hydrolyse carbohydrates, lipids, proteins
etc
 Examples are lipase (triacylglycerol acyl
hydrolase, E.C. 3.1.1.3.), amylase, pepsin,
ribonuclease, sucrase, lactase, maltase, choline
esterase, acid and alkaline phosphtases, uriase
etc

(3.1) Acting on Ester bonds-Esterases
• (a) Acetylcholine esterase
Acetylcholine +H2O Choline + Acetic Acid
• (b) Alkaline Phosphatase
Orthophosphoric mono ester+H2O  an Alcohol +H3PO4
G-6-P + H2O D glucose + H3PO4

(3.2) Acting on Glycosyl compound
• Amylases β -galactosidases
(3.3) Acting on Peptide bonds
• Peptidases  Aminopeptidases,
Carboxypeptidases
(3.4)Acting on C-N bonds
• Deamidases Urease (Urea Amido Hydrolase)
Eg: Urea + H2O  CO2 + 2 NH3

(3.5) Acting on Acid Anhydride bonds
(a)Pyrophosphate Phosphorylase (Synthetic Name)
• This enzyme is also called Inorganic pyrophosphatase
(trivial name)
• Pyro PO4 + H2O 2-Ortho phosphate
(b)ATP Phospho Hydrolase (synthetic name)
• ATPase (trivial name)
• ATP + H2O ADP + Orthophosphate

Lyases
 These enzymes remove chemical groups from
substrates by mechanisms other than hydrolysis
 The groups removed may be water, amino
group, carboxyl group etc
 Examples include aldolase B (ketose 1- phosphate
aldehyde lyase, E.C. 4.1.2.7.), enolase, fumarase
histidase, Arginosuccinase, histidine decarboxylase

(4.1)C-C Lyases : Pyruvate Decarboxylase, Aldolase
2 oxo acids an Acid + CO2
(4.2) C-O lyase : Serine Hydrolyase or
Serine Hydratase, fumerase
Serine + H2O Pyruvic Acid+ NH3 + H2O
(4.3) C-N Lyase : Arginosuccinate lyase

Isomerases
 These enzymes catalyse interconversion of
isomers of a compound
 Examples include alanine racemase, triose
phosphate isomerase (D-glyceraldehyde 3-
phosphate ketoisomerase, E.C. 5.3.1.1.),
phosphohexose isomerase, ribose-5-phosphate
ketoisomerase, retinol isomerase etc

• Eg: Racemases (5.1)- methyle malonyl Co A
racemase
• Epimerases (5.2)- triose phophate isomerase
• Cis-trans Isomerases (5.3)- retinine isomerase

Ligases
 These enzymes ligate or bind two compounds
together
 Since the binding occurs by a covalent bond, a
source of energy is required, usually a high-
energy phosphate
 Examples are glutamine synthetase (L- glutamate
ammonia ligase, E.C. 6.3.1.1.), squalene
synthetase, succinate thiokinase, acetyl CoA
carboxylase, Pyruvate carboxylase, Alanyl-t
RNA synthetase, DNA ligases etc

Ligases can join or form
(6.1) C-O bonds 
Tyrosyl S. RNA Synthetase
Leucyl S.RNA Synthetase
Alanyl S.RNA Synthetase
(6.2) C-S bonds 
Acyl CoA synthetase
(6.3) C-N bonds 
GMP Synthetase,
CTP Synthetase

(6.4) C-C bonds 
(i) Pyruvate CO2 Ligase or Pyruvate Carboxylase
ATP +Pyruvate +CO2 + H2O ADP +Oxaloacetate + Ortho P
(ii) Acetyl CoA CO2 Ligase or Acetyl CoA
Carboxylase
ATP + Acetyl CoA + CO2 + H2O ADP + Ortho P + Malonyl CoA

• Synthetase (requires ATP), synthase (no ATP required)
• Phosphatase (uses water to remove phosphoryl group)
• Phosphorylase (uses Pi to break a bond and generate a
phosphorylated product)
• Dehydrogenase (NAD+/FAD is electron acceptor in redox
reaction)
• Oxidase (O2 is acceptor but oxygen atoms are not
incorporated into substrate), oxygenase (one or both
oxygens atoms are incorporated into substrate)

Measurement of Enzyme Activity
 Enzyme catalyzed reactions are highly efficient,
proceeding at 103 to 106 times faster than
uncatalyzed reactions.
 Each enzyme molecule is capable of transforming 100
to 1000 substrate molecules into products per second
 King- Armstrong unit, somogyi units, reitman- Frakel
units, spectrophometric units etc.

Turn over number
 The number of molecules of substrate converted to
product per enzyme molecule per second is called the
turn over number
 katal (kat): one kat denotes the conversion of one
mole substrate per second into the product per
enzyme molecule (mole/sec)
 mkat, μkat etc

International Units (IU) or System International
Units (SI)
• international units (IU) is usually expressed as
one µmol of substrate transformed to product
per minute per milligram of enzymes under
optimal conditions of measurements.
1IU = 60 µkatal
or
1 nkatal = 1.67 IU

SPECIFICITY OF ENZYME ACTION
 Ability of enzyme to discriminate between two substrates.
 Enzymes are highly specific both in the reaction catalyzed
and in their choice of substrates.
 Specificity makes it possible for number of enzymes to
co-exist in cell without interfering in each other’s actions.

Types of Specificity
1. Substrate specificity
2. Reaction specificity
3. Stereo specificity

Substrate Specificity
i. Absolute substrate specificity
ii. Relative substrate specificity
iii. Broad substrate specificity.

Absolute substrate specificity
Certain enzymes will act on only one substrate
and catalyze one reaction, e.g. Glucokinase,
lactase, urease, etc.

Relative substrate specificity
Enzyme acts on more than one substrate.
 Group specificity
 Bond specificity.

Chymotrypsin acts on several proteins by
hydrolyzing peptide bonds attached to aromatic
amino acids.
Trypsin hydrolyzes peptide linkages involving
arginine or lysine.

α-amylase, cleaves α-(1→4) glycosidic bonds
of carbohydrates.
Lipase hydrolyzes ester bonds of lipids.

Broad substrate specificity
 Enzyme acts on more than one structurally
related substrates.
 hexokinase catalyzes the phosphorylation of
more than one kind of hexoses such as glucose,
fructose and mannose.

Reaction Specificity
Enzyme is specific to a particular reaction but
not to substrate (s) and catalyzes only one type
of reaction.

Figure 6.5: Example of reaction specificity.

Stereo Specificity
 L-lactate dehydrogenase will act only on
L-lactic acid and not D-lactic acid.
 L-amino acid oxidase and D-amino acid
oxidase act only on L and D-amino acids.
 Salivary α-amylase acts on the α-1,4
glycoside linkage and is inactive on β-1,4
glycoside bond

Fischer’s Lock and Key Template model
 This model explains that enzymes have a rigid
pre shaped configuration like a lock, and
substrate has a shape complementary to the
lock that is like a key

Fischer’s Lock and Key Template model

Koshlands Induced Fit Model put forward in
1962
 Koshland’s Induced Fit Model
 This model explains on the basis that active site
of an enzyme is flexible
 It undergoes conformational change to attain
final catalytic shape to suit the substrate
molecule

Catalytic or Active Site of Enzymes
 The enzyme proteins are big large sized
molecules as compared to the substrates which
are relatively smaller.
 Only a portion of the enzyme molecule is
involved in the binding of the substrate

 A small portion of the enzyme protein molecule
which actually takes part in catalysis is called
the Active or Catalytic Site.
 Common features-
1) Active site is a small portion of three dimensional
(3º structure) enzyme proteins.
2) It is situated in the crevice/ clefts/ pocket of the
enzyme molecule. Flexible nature

3)To the active site a specific substrate binds. This
binding of substrate depends on the specific
groups or atoms at the active site.
4) Specific groups come out from the linear amino
acid chain. The residues may be far apart in a
linear sequence, but may come together to bring
about catalysis.

5). During binding these groups may realign
themselves to provide the unique conformational
orientation, so as to promote exact fitting of
substrate to the active site.
6). The substrate binds to the Enzyme at the
active site by weak non-Covalent Bonds.
These forces are hydrophobic in nature.

7).The amino acids or groups that directly
participate in making or breaking the bonds
are called Catalytic residues or groups.
8). The active site contains a substrate binding site
and a catalytic site. Sometimes they may be
separate.
9). Lysozyme has 129 amino acids. Active site is
contributed by 35, 52, 62, 63 and 101 amino
acids.

Allosteric Enzymes
 Some enzymes possess a site, in addition to the
substrate site, known as the allosteric site
 Binding of an allosteric molecule to the
allosteric site affects the conformation of the
substrate site
 Such enzymes are termed as allosteric enzymes

 The allosteric molecule (effector or modifier or
regulator) may facilitate the conformational
change required for substrate binding
 Such regulators are known as allosteric
activators (positive modifiers)
 An example is N-acetylglutamate which is an
allosteric activator of carbamoyl phosphate
synthetase (mitochondrial)

 Some allosteric regulators prevent the
conformational change required for the
binding of the substrate

Such regulators are known as allosteric inhibitors (negative
modifiers)

 The enzymes subject to allosteric inhibition are
generally present in the beginning of long
metabolic pathways
 The allosteric inhibitor is generally the product of
the pathway
 The allosteric enzyme regulates the rate of
formation of the product

 If the product is not being utilised and
accumulates, it inhibits the allosteric enzyme,
and further synthesis of the product is stopped
 When the concentration of the product
decreases, it dissociates from the allosteric
enzyme, and the inhibition is relieved
S I I
1 2
I I P
3 4
4
E 1
E 2
E 3
E 4
E 5
6

Should know about:
 Transition state
 Ground state
 Activation energy
 Enzymes act by reducing the activation energies
 Binding energy
How do enzymes work?

Enzymes
Lower a
Reaction’s
Activation
Energy

Enzymes Affect Reaction Rates, Not Equilibria.
Change
in Free
Energy
(▲G)
The free energy of
reaction, ▲G,
remains unchanged
in the presence of
enzyme, so the
relative amounts of
reactants & products
at equilibrium are
unchanged.

Enzymes reduce the magnitude of activation energy.
Enzymes accelerate reactions by facilitating the formation
of the transition state, which is
* a state in which reactants are in the state of
highest free energy during a chemical reaction.
* the least stable and
* a transitory molecular structure that is no
longer the substrate but is not yet the product

Activation energy:
 the energy required to convert all molecules of a
reacting substance from the ground state to the
transition state.
 It is the difference between the free energy of the
transition state and of the reactants.

Binding Energy:
 The Free energy released in the formation of a large
number of weak interactions between the enzyme and
the substrate.
 Binding energy is a major source of free energy used
by enzymes to lower the activation energies of
reactions.

Factors Affecting the Rates of Enzyme-catalysed
Reactions
1. Enzyme concentration
2. Substrate concentration
3. Coenzyme concentration
4. Product concentration
5. Temperature
6. pH
7. Effect of Activators and inhibitors
8. Effect of ultraviolet (UV) radiation

Enzyme concentration
 An enzyme catalyses a reaction by forming the
enzyme-substrate complex which dissociates into
the enzyme and the product
 The enzyme may be considered to take part in
the reaction as a reactant though it is
regenerated in its original form at the end of the
reaction
E + S E S E + P

 The rate of the initial reaction leading to the
formation of ES is directly proportional to the
product of molar concentrations of E and S
Rate of formation of ES  [E] [S]
 Similarly, the rate of the second reaction leading
to the formation of E and P is directly proportional
to the molar concentration of ES
Rate of formation of E and P  [ES]

 Therefore, the rate of formation of the product i.e.
the rate of the overall reaction is proportional to
the enzyme concentration provided that enough
substrate is available to combine with the enzyme

Substrate concentration
 Just as the rate of the reaction is proportional to
enzyme concentration, theoretically it should be
proportional to substrate concentration also
provided that enough enzyme is available to bind
the substrate
 However, the availability of enzymes in the cells
is limited whereas the concentration of
substrates can vary over a wide range

 When the substrate concentration rises, initially
there is a proportionate increase in the velocity
of the reaction but later the rise in velocity
becomes slower until a maximum velocity (Vmax)
is reached

 At Vmax, all the enzyme molecules are saturated
with the substrate, and no further increase in
velocity is possible even if the substrate
concentration goes on increasing
 The substrate concentration at which the velocity
is half of Vmax is known as the Michaelis constant
(Km) of the enzyme

 The relationship between the velocity of the
reaction and the substrate concentration can be
expressed by Michaelis-Menten equation,
which is:
Km = Michaelis-Menten (or Brig’s and Haldane’s)
constant
[ ]
[ ]
max
V S
v
Km S
=
+
.

 When the substrate concentration is exactly
equal to Km, the sum of Km and [S] may be
taken as 2 [S]
 The equation may be rewritten as:
2
[ ]
max
V S
vmax
Km + [S]
= .
[ ]
[ ]
max
V S
v
Km S
=
+
.

2Vmax. [S]
Km+ [S] = Vmax
Km+ [S] = 2[S]
Km = [S]
Thus, when the substrate concentration is
equal to Km, the velocity is half of Vmax

Determination of Km
 Determination of Km is important in the study of
enzyme kinetics, assay of enzyme activity and
evaluation of enzyme inhibitors
 Km or the Michaelis-menten constant is substrate
concentration (moles/ L) to produce half maximum
velocity in enzyme catalysed reaction
 It is indicate the half of the enzyme molecules
(50%) are bound with substrate molecules
when substrate concentration equals the Km
value

Km and affinity of Substrate
 Km indicates the affinity of the substrate towards
the enzyme and is inversely proportional to the
affinity.
 Km α 1
affinity
 Low Km Higher affinity
 Higher Km Lower affinity

Importance of Km
 It is indicate the half of the enzyme molecules (50%)
are bound with substrate molecules when substrate
concentration equals the Km value
 It indicates the degree of affinity of an enzymes for a
particular substrate.
 Km is neither influenced by enzyme concentration nor
by noncompetitive inhibitors
 It is altered by competitive inhibitors; allosteric
modulators, pH, temperature and substrate
concentration.

 Lineweaver and Burk plot is a simple method in
which velocity is measured at a small number
(5- 6) of substrate concentrations, and a graph
is plotted between the reciprocal of v and the
reciprocal of [S]
 The 1/v versus 1/ [S] plot is known as
Lineweaver-Burk plot or double reciprocal plot

 Michaelis-Menten equation is inverted
 This is the equation for a straight line i.e. y = ax+b
where x (x-axis) is 1/[S], y (y-axis) is 1/v , a (slope
of the line) is Km/Vmax and b (y-intercept) is 1/Vmax
[ ]
[ ]
max
Km S
1
V V S
+
=
.
or
[ ]
[ ]
[ ]
max
S
1 Km
=
max
+
. S
V .
or
[ ]
max
1 Km 1 1
=  +
max
V V S V
V V S

 Thus, the x-intercept i.e. the value of 1/[S] at the
x-intercept gives the value of 1/Km, and the
reciprocal of this will be the Km
1
1
1
1
Vmax
[ S ]
Km
v

 Allosteric enzymes do not follow Michaelis-
Menten equation
 The v versus [S] plot of allosteric enzymes is
sigmoidal showing co-operative binding of the
substrate to the enzyme
V

Positive effectors shift the plot to the left,
and negative effectors shift it to the right
V
 Kinetics of allosteric enzymes follow the Hill equation

Effect of product concentration
When product concentration is increased, the
velocity of the enzyme is slowed or even stopped.
Reaction may even be reversed in a reversible
reaction.
In a metabolic pathway, accumulation of a product
intermediate can gradually inhibit the activity of
preceding enzymes.

Coenzyme concentration
 If a coenzyme is required in the reaction, the
concentration of coenzyme can also affect the
velocity of the reaction
 Some coenzymes are very tightly bound to the
apoenzyme, and form an integral part of the
holoenzyme molecule
 Other coenzymes act as co-substrates in the
reaction

 If the coenzyme is an integral part of the
enzyme, the effect of coenzyme concentration
will be identical to that of the enzyme
concentration
 If the coenzyme act as a second substrate, the
effect of coenzyme concentration is similar to
that of the substrate concentration

Temperature
 If the velocity of a reaction is measured at
different temperatures, and a curve is plotted
between velocity and temperature, a bell-
shaped curve is obtained
 Initially, when the temperature rises, the velocity
increases due to increase in the kinetic energy
of the reactants

 A further rise in temperature leads to progressive
denaturation of the enzyme, and the velocity
begins to decrease until the reaction practically
stops when the enzyme is completely denatured
 The temperature at which the velocity is the
highest is known as the optimum temperature of
the enzyme
 For all human enzymes, the optimum
temperature is 37°C

 In the initial part of the curve, the number of
times the velocity increases when the
temperature rises by 10°C is known as the
temperature coefficient (Q10) of the enzyme
 For most of the enzymes, the temperature
coefficient is two
 This means that the velocity is doubled when
the temperatures rises by 10°C

pH
 If the velocity of the reaction is determined at
different pH levels, and the velocity is plotted as
a function of pH, a bell-shaped curve is obtained
 A change in pH alters the electrical charges on
the enzyme molecules, and often on the
substrate molecules as well
 This may affect the binding of the substrate to the
enzyme or the catalytic activity of the enzyme or
both

 At an optimum pH, the velocity of the reaction is
the highest as the electrical charges on the
enzyme and the substrate are the most suitable
for enzyme-substrate binding and catalysis
 As we move away from the optimum pH, the
velocity of the reaction decreases
 At extremely low or high pH, the enzyme may be
denatured
 The optimum pH is different for different enzymes

 Usually enzymes have optimum pH around
neutral pH i.e. pH 6 to 8 (optimum pH for pepsin is
pH 1 to 2, while that of alkaline phosphatase is
pH 9 to 10).

Effect of Activators and inhibitors
 Activators like metal ions (Mg2+, Mn2+ Zn2+, Ca2+,
Co2+, Cu2+, Na+, K+ etc) increase the activity of
the enzymes. Magnesium ion is an activator for
kinases and chloride ion activates salivary
amylase.
Various inhibitors both reversible and irreversible,
bind to enzymes and decrease their activity.

• Metal-activated enzymes---
• e.g. ATPase (Mg2+ and Ca2+), Enolase (Mg2+)
• Metalloenzyme---
• eg. Alcohol dehydrogenase, Aarbonic anhydrase,
Alkaline phosphatase, Aarboxypeptidase and
Aldolase (zinc)
• Phenol oxidase (copper);
• Pyruvate oxidase (manganese);
• Xanthine oxidase (molybdenum);
• Cytochrome oxidase (iron and copper).

Effect of ultraviolet (UV) radiation
Exposure to UV rays, X-rays, and γ-rays causes
peroxides formation, which oxidize the enzymes
and inactivate them.
UV rays inhibit activity of salivary amylase.
Radiation can also damage DNA and impair
synthesis of enzymes.

 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.

 Inhibition may be reversible , where the inhibitor
does not react covalently with the enzyme
(reversible inhibition).
 Some agents react covalently with the functional
groups of enzymes resulting in non-competitive
irreversible inhibition.

Inhibition
Reversible Irreversible Allosteric
Competitive Non-Competitive
Classification of Inhibition

Competitive Inhibition
 The inhibitor (l) which closely resembles the real
substrate( S) is regarded as a substrate analogue.
 The inhibitor competes with substrate and binds at
the active site of the enzyme but does not undergo
any catalysis.
 Competitive inhibitor holds the active site, the
enzyme is not available for the substrate to bind.
Reversible inhibition

A. Effect of a competitive inhibitor on the reaction
velocity (vo) versus substrate ([S]) plot. B. Lineweaver-
Burk plot of competitive inhibition of an enzyme.

Competitive Inhibition
 Affinity Decreases
 I Decreases as Km increases
Km
 I Remains the same (unchanged)
Vmax
Efficiency Remains the same

Examples of Competitive Inhibitors
S.N Enzyme Substrate Inhibitor
1. LDH Lactate Oxamate
2. Aconitase Cis-Aconitate Trans-
Aconitate
3. Succinate
Dehydrogenase
Succinate Malonate,
Oxalate,
Glutarate,
Adipicate
4. H.M.G. Co A
reductase
HMG Co A Lovastatin,
Mevastatin,
Compactin
5. Dihydrofolate
reductase
7,8 dihydrofolate Aminopterin,
Amethopterin,
Methotrexate

6. Xanthine Oxidase Hypoxanthine,
Xanthine
Allopurinol
used in Gout
Treatment
7. Acetylcholine
esterase
Acetylcholine Succinyle
choline
8. Vitamin K epoxide
reductase
Vitamin K Dicumarol
9. Dihydropteroate
synthase
PABA (Para
amino benzoic
acid)
Sulfonilamide
10.
Adenylo succinate
Synthetase
6-Mercapto
Purine

11. Acetylcholine
esterase
Use in Myasthenia
gravis
Acetylcholine Neostigmine,
Physostigmine
12. DOPA Decarboxylase
Use in Hypertension
DOPA Alpha methyl
DOPA
13. Transpeptidase inhibit bacterial
cell wall
synthesis.
Penicillin,
Amoxicillin
14. Dihydrofolate
reductase in bacteria
7,8 dihydrofolate Trimethoprim
15. Dihydrofolate
reductase in malarial
7,8 dihydrofolate Pyrimethamine

16. Thymidylate
synthase
Thymidine 5- flurouracil
17. Alcoholic
dehydrogenase
Methanol Ethanol
18. Angiotensin-
Converting enzyme
(ACE)
Angiotensin I Captopril,
Enalapril,
Lisinopril
(hypertension)
19. Thymidine kinase Thymidine Isoxuridine
(Antiviral drug)
21. Monoamine oxidase Epinephrine,
Norepinephrine
Ephedrine,
Amphetamine
20. Inhibits the synthesis of
Prostaglandin and thromboxane
Aspirin

Examples of Competitive Inhibitors used
as Drugs Clinically
 Allopurinol ---- Used in the treatment of Gout
Xanthine Oxidase
Hypoxanthine Uric Acid
Allopurinol
N
HN
O
||
C
C
C
HC
N
H
H
C
N
N
HN
O
||
C
C
C
HC
N
H
N
CH
Hypoxanthine Allopurinol
Hypoxanthine
Xanthine
oxidase
Uric acid
Xanthine
Xanthine
oxidase
Alloxanthine

Sulfonilamide
 Used as antibacterial agents. Similar in structure to
PABA (Dihydrobiopteroate synthase)
 For Folate synthesis
PABA is essential
Sulfonilamide
Needed for Bacterial Growth

Methotrexate, Amethopterin and
Aminopterin
 Methotrexate is 4-amino N10 methyl folic acid.
 Used in cancer therapy
 Methotrexate resembles folic acid it competitively
inhibits “dihydrofolate reductase”
 Prevents the formation of FH4
 DNA Synthesis is inhibited
 Methotrexate is toxic producing symptoms like lose of
hair, vomiting, diarrhea

Folic acid
Amethopterin
H N
2 N
N
|
OH
1
2
3
4
N
5
6
7
8
N
9 10
CH — N —
2 — C — N — CH
| |
H COOH
COOH
|
CH2
|
CH2
|
O
||
H
|
Pteridine para-Amino-
benzoic acid
Glutamic
acid
Pteroylgutamic acid (folic acid)
H N
2 N
N
|
OH
1
2
3
4
N
5
6
7
8
N
9 10
CH — N —
2 — C — N — CH
| |
H COOH
COOH
|
CH2
|
CH2
|
O
||
H
|
Pteridine para-Amino-
benzoic acid
Glutamic
acid
Pteroylgutamic acid (folic acid)
C
3

Dihydrofolate + NADPH + H +
Tetrahydrofolate + NADP +
Dihydrofolate
reductase
4
Folate + NADPH + H
Dihydrofolate
reductase
4
+

MAO Inhibitors
 MAO inhibitors are Ephedrine and Amphetamine
 Enzyme Mono Amine Oxidase oxidizes Epinephrine
and Nor-epinephrine
 MAO inhibitors competitively inhibit MAO, prolong
action of presser amines.
 Useful for elevating catecholamine level

Physostigmine and Neostigmine
 Physostigmine is Acetylcholine esterase inhibitor
 Acetylcholine Acetate + Choline
 This drug prevents destruction of Acetylcholine,
 Continued presence of Acetylcholine in post
synaptic regions prolong neural impulse.
 used as drugs in which the concentration of
acetylcholine needs to be increased e.g. Myasthenia
gravis, an autoimmune disorder

Mevastatin, Lovastatin, Pravastatin
(Pravachol) and Atorvastatin (Liptor)
 These resemble HMG CoA in structure, and are
competitive inhibitors of HMG CoA reductase
HMG CoA
Mevalonate
HMG CoA
reductase
4
Cholesterol

Aspirin
 It is used as an anti-inflammatory agent.
 Aspirin acetylates the serine residue present at the
active site of cyclooxygenase enzyme, which is
involved in prostaglandin synthesis.
 Inhibition of prostaglandin synthesis subsides
inflammation.

Dicumarol
• Dicumarol is used as an anticoagulant
• It competitively inhibits Vitamin K

Non-Competitive Inhibition
 No competition between the Inhibitor and substrate.
 These inhibitors do not resemble the substrate and
bind to a site away from the active site.
 Enzyme inhibitor has normal affinity for the substrate
but produce products at a decreased rate.

A. Effect of a noncompetitive inhibitor on the reaction
velocity (vo) versus substrate ([S]) plot. B. Lineweaver-
Burk plot of noncompetitive inhibition of an enzyme.

Non-Competitive Inhibition
 Affinity Remains the same
 Efficiency decreases
 1/ Km remains the same as substrate
concentration has no effect on the inhibitor
 1/ Vmax Increases as V has a decrease
 For non-competitive inhibition, the Km value is
unchanged while Vmax is lowered

 Non-competitive inhibition can be reversed if the
inhibitor can be removed without affecting the enzyme
activity.
 eg: Enzymes with –SH groups bind to heavy metals
like Hg , Pb, Ag etc. resulting in non-competitive
inhibition.
 It can be reversed not by high levels of substrate but
by increasing –SH in the medium.

Irreversible inhibition
 Inhibitors bind covalently with the enzymes inactive
them
 Irreversible
 Toxic poisonous subtances
 eg. Iodoacetate bind (SH- group) with papain and
glyceraldehyde 3 phosphate dehydrogenase
 Diisopropyl fluorophosphate (DFP) bind serine
proteases, acetylecholine esterase
 Disulfiram bind ALD

 Heavy metal ions eg. Ag, Hg, also act as
irreversible noncompetitive inhibition
 Fluoride (NaF) inhibits glycolytic enzyme by
replacing Mg and Mn
 BAL (British anti Lewesite)/ Dimercaprol used as
antidote for heavy metal poisoning
 Ferrochelatase (heme synthesis enzyme)
inhibited by lead

Allosteric inhibition
 Allosteric enzymes are oligomeric (multi subunit)
enzymes, which contain an active site, and a separate
allosteric site for regulation of enzyme activity.
 Negative modifier or inhibitor is not a substrate
analogue. It binds non-covalently and reversibly to
the allosteric site that causes a conformational
change in the enzyme, which results in decreased
activity of the enzyme.

This type of enzyme inhibition is known as
allosteric inhibition and is partially reversible by
adding excess amount of substrate.
Allosteric enzymes show either positive or negative
co-operativity in substrate binding.
Regulatory enzymes are allosteric enzymes, which
can undergo feedback inhibition or end product
inhibition.

Positive effectors shift the plot to the left,
and negative effectors shift it to the right
 Kinetics of allosteric enzymes follow the Hill equation

S.
N.
ENZYME Allosteric
Inhibitor
Allosteric
activator
1 HMG Co A -reductase Cholesterol
2 Phosphofructokinase ATP,
Citrate
AMP,
F2,6,P
3 Pyruvate Carboxylase ADP Acetyl Co
A
4 Acetyl CoA
Carboxylase
AcylCoA Citrate
5 Citrate Synthase ATP

S.N. ENZYME Allosteric
Inhibitor
Allosteric
activator
6 Carbamyl Phosphate
Synthetase- I
N-Acetyl
Glutamate
7 Carbamyl
PhosphateSynthetase-
II
UTP
8 Aspartate
Transcarbamylase
CTP ATP

 Many of these molecules are very effective
drugs, because they are targeted specifically for
a certain enzyme and kill the enzymes for good.
 This inhibitors kill the enzyme for good, but
since they also 'die' in the process, they are
called suicide or mechanism-based
inhibitors.
Suicide Inhibition

Suicide inhibition
 It is a type of irreversible inhibition
 The inhibitor makes use of an enzyme own reaction
mechanism to inactivate it
 In suicide inhibition, the structural analogue is
converted to a more effective inhibitor with the help
of the enzyme to be inhibited.
 This new product binds to the enzyme and inhibits
further reaction.

1. Allopurinol
 Allopurinol a competitive inhibitor for enzyme
xanthine oxidase
 When it comes in contact with the enzyme it is
oxidized by xanthine oxidase to alloxanthine which
is a stronger irreversible inhibitor of the enzyme
Xanthine Oxidase
Hypoxanthine Uric Acid
Allopurinol

2. Anti-inflammatory Action of Aspirin
 Membrane bound phospholipids are broken down
first to Arichidonic acid (by phospholipases)
Cyclooxygenase
Arichidonic Acid Prostaglandins
 Aspirin acetylates a serine residue in the active
center of cyclooxygenase, inhibiting prostaglandin
synthesis and reducing inflammation

3. 5-fluorouracil
 5- fluorouracil is a anticancer drug
5- fluorouracil
enzyme of salvage pathway
fluorodeoxyuridylate that inhibits thymidylate synthase
4. Ferrochelatase (heme synthesis
enzyme) inhibited by lead

5. Difluromethyl ornithine against sleeping
sickness Trypanosomiasis
 Ornithine Decarboxylase converts Ornithine to
putrescince a polyamine
 When this enzyme ODC in Trypanosoma (parasite)
is inhibited, multiplication of the parasite is arrested.
 Difluoromethyl ornithine (DFMO) is initially inert.
Binding with the enzyme it forms an irreversible
covalent complex with co-enzyme Pyridoxal PO4
(vit. B6) and amino acid residues
 used in the treatment of trypanosomiasis
(sleeping sickness)

Inhibitor type Binding site on the enzyme Effect on enzyme
kinetics
Competitive
inhibitor
Competes with substrate for binding to
the active site. Inhibition is reversible by
high substrate concentrations.
Vmax is unchanged
Km is increased.
Noncompetitive
inhibitor
Binds to a site other than active site.
Hence it can bind both to the E or ES
complex. ESI complex cannot form
products. Therefore inhibition is not
overcome even at high substrate
concentration.
Vmax is proportionally
decreased to inhibitor
concentration.
Km appears unaltered.
Uncompetitive
inhibitor
Binds only to the ES complex, at a site
other than the active site. The substrate
binding to active site alters the enzyme
structure, such that the inhibitor site is
made available for inhibitor binding.
Inhibition is not reversible even at high
substrate concentration.
Apparent Vmax is
decreased.
Km is also decreased.

SERUM ENZYMES DISEASES
Amylase(80-180 SI/dl) Acute pancreatitis, mumps, diabetic
ketoacidosis.
SGPT(3-40 IU/L) Liver diseases (hepatitis)
SGOT(4-45 IU/L) Myocardial Infarction
ALP(3-13 KAunits/dl) Rickets, Osteitis, Obstructive jaundice
ACP(0.5-4 KAunits/dl) Carcinoma of prostate gland.
LDH(50-200 IU/L) Heart attacks, liver diseases.
CPK(10-50 IU/L) MI (earliest marker)
Aldolase(2-6 IU/L) Muscular Dystrophies
5’-Nucleotidase(2-15 IU/L) Hepatitis
γ- Glutamyl Transpeptidase
(GGT)(5-40 IU/L)
Alcoholism.

Enzymes of Diagnostic Importance
 A large number of enzymes are synthesised in
the cells
 They are continuously released into circulation in
small amounts as a result of the normal wear
and tear of cells
 They are removed from circulation by
degradation or excretion

 These enzymes are normally present in
circulation in minute concentrations
 The circulating enzymes may be divided into
two types:
A. Functional plasma enzymes or plasma-
specific enzymes
B. Non-functional plasma enzymes or non-
plasma- specific enzymes

Functional plasma enzymes or plasma-specific
enzymes
• These enzymes are purposely secreted into
circulation to perform specific catalytic functions
• These include lipoprotein lipase, blood
coagulation factors, complement proteins,
renin, cholinesterse, ceruloplamin etc
• Deficiency of ceruloplasmin in Wilson's disease
• 1IU = 60 µ kat

Non-functional plasma enzymes or non-plasma-
specific enzymes
• These enzymes do not perform their catalytic
functions in plasma
• These are the intracellular enzymes which
enter the circulation when the cells in which they
are synthesised disintegrate

• The digestive enzymes of the gastrointestinal
tract (e.g. amylase, pepsin, trypsin, lipase etc.)
present in the plasma are known as secretory
enzymes.
• Plasma enzymes associated with metabolism of
the cell are collectively referred to as constitutive
enzymes (e.g. lactate dehydrogenase,
transaminases, acid and alkaline phosphatases,
creatine phosphokinase)

 When breakdown of cells is occurring at
normal rate, these enzymes are present in
plasma in very low concentrations
 If the rate of destruction of cells increases due to
some pathological condition- increased cell
turnover, abnormal cell proliferation of cell
(neoplasia) etc, these enzymes will be released
into circulation in large amounts, and their
concentrations in plasma will rise many times
above normal

The following plasma enzymes have become
established diagnostic tools:
1. Lactate dehydrogenase (LDH)
2. Transaminases (SGOT and SGPT)
3. Creatine kinase (CK) /Creatine phospho kinase (CPK)
4. Gamma glutamyl transpeptidase (GGT)
5. Alkaline phosphatase (ALP)
6. Acid phosphatase (ACP)
7. Amylase
8. Lipase
9. Ceruloplasmin

Lactate dehydrogenase (LDH)
 This enzyme catalyses the interconversion of
pyruvate and lactate
 Its tissue distribution is very wide
 However, its concentration is much higher in
myocardium, muscles and liver than in other
tissues

 Therefore, plasma LDH rises in myocardial
infarction, viral hepatitis and muscle injuries
 In myocardial infarction, the rise begins 24 hours
after the episode of infarction, the peak value is
reached in about three days, and the level
returns to normal in about a week
 The normal pattern of LDH isoenzymes is
LDH2 >LDH1 >LDH3 >LDH4 >LDH5

 LDH 1 is also seen in germ cell tumors
(Seminoma of testis and dysgerminoma of
ovary)
 LDH 3 is also seen in pulmonary embolism
 LDH 4 in muscular dystrophy
 LDH 5 in Liver diseases

Transaminases
 The two most important transaminases are
glutamate oxaloacetate transaminase (GOT)
and glutamate pyruvate transaminase (GPT)
 These are also known as aspartate
aminotransferase (AST) and alanine
aminotransferase (ALT) respectively
 These are present in high concentrations in
myocardium, liver and muscles

 Therefore, their plasma levels are raised in
myocardial infarction, viral hepatitis and muscle
injuries
 Concentration of GOT is higher than that of GPT
in myocardium while the situation is reverse in
liver
 Therefore, the rise in plasma GOT is more
pronounced in myocardial infarction and that in
GPT is more pronounced in viral hepatitis

Creatine kinase (CK)
 It is also known as creatine phosphokinase
(CPK), and catalyses the following reaction:
 CK is present in myocardium, muscles and brain
Creatine + ATP Creatine ~ P + ADP

 Plasma CK rises in myocardial infarction,
myopathies and muscle injuries
 Plasma CK2 (MB) is a more specific and early
indicator of myocardial infarction than LDH1 and
GOT
 It begins to rise within 3-6 hours of occurrence
of infarction, reaches its peak in 24 hours, and
returns to normal in about three days
 Also increased in acute cerebrovascular accidents

1 2 3 4 5 6 7
0
Enzyme
level
Upper limit
of normal


Days
CK GOT LDH

Enzyme markers of myocardial infarction
CPK, SGOT and LDH are released from myocardium
after myocardial infarction, and are useful in diagnosis
Begins to
rise in
Reaches
peak in
Returns to
normal in
CPK 3-6 hrs 24 hs 3 days
SGOT after CPK 48 hs 4 - 5 days
LDH 24 hrs 3 days 7 days

Non-enzyme markers of myocardial infarction
Apart from LDH, GOT and CPK, some non-enzyme
proteins are also released from myocardium after
myocardial infarction, and are useful in diagnosis
Begins to
rise in
Reaches
peak in
Returns to
normal in
Specificity
Myoglobin 1-3 hrs 4-6 hs 18-24 hrs Low
Cardiac troponin T 4-6 hrs 18-36 hs 5-15days Low
Cardiac troponin I 4-6 hrs 12-24 hs 5-10days High

Cardiac troponin T Tropomysin binding element
Cardiac troponin I Inhibitory element of actinomycin
ATPase

Gamma glutamyl transpeptidase (GGT)
 This enzyme catalyses the transfer of the
gamma-glutamyl residue of glutathione to
other substrates
 Its plasma level increases in most of the liver
diseases, and is an early indicator of alcoholic
hepatitis or fatty liver disease

Alkaline phosphatase (ALP)
 This is a group of enzymes that hydrolyse
organic phosphate esters at an alkaline pH
 ALP is released in circulation mainly from
bones and liver
 Smaller amounts come from intestines and
placenta
 Liver excretes ALP in bile

 The maximum elevation of plasma ALP occurs in
obstructive jaundice and Bone disease
 Smaller elevations occur in viral
hepatitis, rickets, hyperparathyroidism,
osteosarcoma, bony metastases etc.

Acid phosphatase (ACP)
 This enzyme hydrolyses organic phosphate
esters at an acidic pH
 The main source of ACP is the prostate gland
 Plasma ACP is elevated in metastatic
carcinoma of prostate

Amylase
 This is a digestive enzyme, synthesised in the
pancreas and the parotid gland
 Sharp elevation of plasma amylase occurs in
acute pancreatitis
 A smaller elevation occurs in acute parotitis
(mumps)

Lipase
 This lipolytic enzyme is released into circulation
from the pancreas
 Plasma lipase rises in acute pancreatitis

Ceruloplasmin
 This is a copper-containing protein having
ferroxidase activity
 It is absent or greatly decreased in plasma in
an inherited disorder, Wilson’s disease
(hepatolenticular degeneration)

ISOENZYMES
 Some enzymes exist in multiple molecular
forms which catalyse the same reaction but
differ in their physical and chemical properties
- structure, electrophoretic, chromatographic
and immunological properties, Km and Vmax
values, pH optimum, relative susceptibility to
inhibitors and degree of denaturation
 The multiple forms of an enzyme catalysing
the same reaction are isoenzymes or isozymes

 Isoenzymes possess quaternary structure, and
are made up of two or more different subunits. The
subunits have slightly different primary structures
 The isoenzymes can be separated from each
other by electrophoretic, chromatographic or
immunochemical techniques
 Separation and quantitation of isoenzymes can
give information of great diagnostic importance
as the tissue distribution of isoenzymes is quite
specific

 Several enzymes exist in the form of
isoenzymes
 The following have been found to be of
particular diagnostic importance:
• Lactate dehydrogenase
• Creatine kinase/ Creatine phosphokinase
• Alkaline phosphatase

Lactate dehydrogenase
 Lactate dehydrogenase was the first enzyme
shown to exist in the form of five isoenzymes
by Markert (1956)
 The enzyme is a tetramer made up of two types
of subunits – H and M
 L-lactate-NAD+ oxidoreductase (E.C.1.1.1.27)
 Separated by electrophoresis (cellulose or
starch gel or agarose gel).

 LDH1 has more positive charge and fastest in
electrophoretic mobility while LDH5 is the slowest.
 LDH1 (H4) is predominantly found in heart muscle and
is inhibited by pyruvate. Pyruvate is not converted to
lactate in cardiac muscle but is converted to acetyl
CoA which enters citric acid cycle.
 LDH5 (M4) is mostly present in liver and skeletal
muscle, inhibition of this enzyme by pyruvate is
minimal, hence pyruvate is converted to lactate.
H subunit- Acidic nature M subunit- Basic nature

 These subunits can form five different tetramers
(isoenzymes):
i. HHHH or LD1 or LDH1 25% Fastest
ii. HHHM or LD2 or LDH2 35% Faster
iii. HHMM or LD3 or LDH3 27% Fast
iv. HMMM or LD4 or LDH4 8% Slow
v. MMMM or LD5 or LDH5 5% Slowest
LDH1 LDH2 LDH3
LDH4 LDH5

 The normal pattern of LDH isoenzymes is
 The predominant isoenzymes in myocardium are
LDH1 and LDH2
 In myocardial infarction, the rise in LDH1 is
greater than that in LDH2

 Therefore, plasma LDH pattern becomes
 Flipped pattern or ratio – normally LDH1 and
LDH2 ratio is less than one but in MI ratio is
more than one
 The predominant isoenzyme in liver is LDH5
which is raised in viral hepatitis

 Total serum LDH is frequently elevated in neoplastic
diseases
 An increase in LDH5 seen in breast carcinoma,
malignancies of CNS, prostatic carcinoma
 In leukemia, LDH2 and LDH3 are increased
 Malignant tumors of testes and ovary show rise of
LDH2, LDH3, and LDH4

Creatine kinase
 Creatine kinase is a dimer made up of two types
of subunits – B and M
 Three different dimers (isoenzymes) can be
formed from these two subunits:
i. BB or CK1 or CK-BB
ii. MB or CK2 or CK-MB
iii. MM or CK3 or CK-MM
B subunit M subunit

 The major isoenzyme in myocardium is CK-MB
 CK-MB is normally less than 3% of total serum
CK. CK-MB is commonly measured by
immunoinhibition
CK-BB
CK-MB
CK-MM

Alkaline phosphatase
 Bone, liver, intestine and placenta form different
isoenzymes of ALP which can be separated by
electrophoresis
 The bone isoenzyme is raised in plasma in bone
diseases and the liver isoenzyme in liver
diseases.

Differentiate by carbohydrate content (sialic acid
residues
 Bone isoenzyme: Increases due to osteoblastic
activity
 In pregnancy: During last six weeks of pregnancy,
placental isoenzyme of ALP increases. It is
inhibited by L-phenylalanine

 Atypical ALP-isoenzymes-“oncogenic markers”
 Regan isoenzyme ALP- Highest incidence of
positivity found in cancers of ovary and uterus. It is
inhibited by L-phenyl alanine
 Nagao isoenzyme ALP- carcinoma of pleural
surfaces and adenocarcinoma of pancreas and
bile duct. It can be inhibited by L-leucine.

ENZYMES AS LABORATORY TOOLS
 Many enzymes are used as tools in diagnostic
and research laboratories:
 Glucose oxidase and peroxidase are
routinely used for measurement of glucose
concentration
 Hexokinase and glucose-6-phosphate
dehydrogenase are used in another method for
measurement of glucose concentration

 Cholesterol esterase, cholesterol oxidase and
peroxidase are used for measuring cholesterol
concentration
 Lipase, glycerol kinase, glycerol phosphate
oxidase and peroxidase are used for measuring
triglyceride concentration
 Urease is used for measurement of urea
concentration

 Uricase is used for measuring uric acid
concentration
 Alkaline phosphatase / Horse radish
Peroxidase are used to label antibodies in
ELISA
 A number of enzymes are used in recombinant
DNA technology e.g. restriction endonucleases,
DNA ligase, terminal transferase, S1 nuclease,
reverse transcriptase, Taq DNA polymerase etc

ENZYMES AS DRUGS
 Some human, animal, plant and microbial
enzymes are used as drugs also
 Streptokinase, urokinase and tissue
plasminogen activator are used as
thrombolytic drugs to clear blockage of
blood vessels e.g. in myocardial infarction

 Some digestive enzymes e.g. diastase, papain
(Atiinlammatory), pepsin, chymotrypsin etc
are used to aid digestion
 α1- antitrypsin is used in treatment of
emphysema
 Asparaginase is used in the chemotherapy of
leukaemia

Enzymes for UNDERGRADUATES AS PER RECENT NMC CURICULLUMRRI

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