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Enzymes
The biological catalysts
Dr. Sangeeta Khyalia
M.D. (Biochemistry)
Jaipur, India
A vast multitude of chemical reactions
occur in living organisms
It is these reactions that keep the
organism going
These reactions would occur at extremely
low velocities in the absence of catalysts
Common catalysts used in non-living
systems are:
Acids Alkalis Metals
These are not suitable for living
organisms because of their:
Toxicity Lack of specificity
Biological catalysts should be:
Safe (non-toxic)
Specific (generally catalyzing
only one reaction)
Capable of adjusting their
catalytic activity
All these properties are present in
enzymes
Enzymes were first discovered in yeast
(enzyme means ‘in yeast’)
They were later found in other living
organisms as well
They could catalyze reactions outside
the living organisms also
Chemically, all enzymes were found to be
proteins
Definition
Enzymes are protein catalysts that catalyse
chemical reactions in biological systems
But this definition is not entirely correct
Some RNA molecules (ribozymes) have
been found to catalyze some reactions
The reactant on which the enzyme acts is
known as the substrate of the enzyme
The enzyme converts the substrate into a
product or products
Substrate
Enzyme
Product
Enzyme specificity
If an enzyme catalyses a number of
reactions, it will be impossible to
regulate individual reactions
However, this doesn’t happen; the
enzymes are highly specific
Generally, one enzymes catalyses
only one reaction
This is of crucial importance for
regulation of metabolic pathways
However, the degree of specificity
may differ in different enzymes
Enzyme specificity may have
the following orders:
Group specificity
Substrate specificity
Stereo-specificity
Group specificity
Enzyme is specific for a chemical
group or bond but not for the actual
substrate
Group-specific or bond-specific
enzymes are commonly present in
digestive secretions
For example, pepsin is specific for
peptide bond but not for any protein
Thus, a large variety of dietary proteins
can be digested by the same enzyme
Trypsin, chymotrypsin, nucleases, lipases
and glycosidases are other examples
Some group-specific enzymes have a
slightly higher degree of specificity
For example, aminopeptidase
hydrolyses only N-terminal peptide bond
Carboxypeptidase hydrolyses only the
C-terminal peptide bond
Endopeptidases hydrolyse the internal
peptide bonds only
H2N–Phe–Ala–Ser–Cys–Gly–Asp–Arg–Val–Leu–Glu–COOH
Amino-
peptidase
Endo-
peptidase
Carboxy-
peptidase
Most enzymes are specific for a chemical
bond/group as well as the substrate
For example, glucokinase and fructokinase
are substrate-specific enzymes
They transfer a phosphate group from
ATP to one specific substrate
Substrate specificity
Glucose-6-Phosphate
Glucokinase
Glucose
ATP ADP
Fructokinase
Fructose Fructose-1-Phosphate
ATP ADP
Substrate-specific enzymes
Stereo-specificity
Many biomolecules exhibit stereo-
isomerism
Examples are carbohydrates and amino
acids
Enzymes acting on these are specific for
one stereo-isomer
Mammalian enzymes acting on carbo-
hydrates are generally specific for
D-isomers
Those acting on amino acids are
generally specific for L-isomers
Exceptions are racemases which
inter-convert the D- and L-isomers
COOH
I
H2N – C – H
COOH
I
H – C – NH2
Alanine
racemase
I
CH3
L-Alanine
I
CH3
D-Alanine
Stereospecificity – An exception
Coenzymes and cofactors
Some enzymes require a non-protein
substance for their catalytic activity
If the non-protein substance is
organic, it is known as a coenzyme
If the non-protein substance is
inorganic, it is known as a cofactor
In some cases, the coenzyme is an
integral part of the enzyme
In others, its presence is required
during the reaction
The protein portion of an enzyme that
requires a coenzyme is called apoenzyme
Apoenzyme +Coenzyme → Holoenzyme
Apoenzyme combines with coenzyme to
form the active holoenzyme
COENZYME
APOENZYME HOLOENZYME
The coenzymes generally contain
vitamins of B-complex family
Some act as coenzymes by them-
selves e.g. biotin
Others are converted into coenzymes
B-Complex
vitamins that are
converted into
coenzymes are:
Thiamin
Riboflavin
Niacin
Pantothenic acid
Pyridoxine
Folic acid
Vitamin B12
Coenzymes are generally
required in group transfer
reactions such as:
Oxidation-reduction
Transamination
Phosphorylation
Coenzymes can be divided into
two groups:
Coenzymes
involved
in transfer of
hydrogen
Coenzymes
involved in transfer
of groups other
than hydrogen
Coenzymes
involved in
transfer of
hydrogen:
Flavin mononucleotide (FMN)
Flavin adenine dinucleotide (FAD)
Nicotinamide adenine dinucleotide
(NAD+)
Nicotinamide adenine dinucleotide
phosphate (NADP+)
Lipoic acid
Coenzyme Q
Coenzymes
involved in
transfer of
groups
other than
hydrogen:
Thiamin pyrophosphate (TPP)
Coenzyme A (Co A)
Pyridoxal phosphate (PLP)
Tetrahydrofolate (H4- Folate)
Cobamides (B12- Coenzymes)
Lipoic acid
Biotin
ATP & similar nucleotides
Role of coenzymes
The enzyme acts upon its substrate, and
converts it into a product
Coenzyme acts as a co-substrate (second
substrate) in group transfer reactions
The coenzyme donates or accepts the
group that is being transferred
EMB-RCG
In the second reaction, the coenzyme NAD+
acts a second substrate and accepts the
hydrogen atoms
In the first reaction, the coenzyme ATP acts
as a second substrate and donates a
phosphate group
CH2‒OH
CH‒OH
CH2‒OH
CH‒OH
CH2‒OH
C =O
CH2‒O‒
CH2‒OH ATP
Glycerol
Glycerol
kinase
ADP CH2‒O‒
Glycerol-3-
phosphate
Dihydroxy-
acetone
phosphate
Glycerol-3-
phosphate
dehydrogenase
NAD+ NADH
+H+
The chemical change in coenzyme is
opposite to that in the substrate
If the substrate loses a chemical group,
the coenzymes accepts it
If the substrate gains a chemical group,
the coenzymes provides it
They act only as carriers, and regain their
original form at the end of the reaction
Pyridoxal phosphate, for example, acts as
a carrier of amino group in transamination
Some coenzymes accept a group from
one substrate and donate it to another
Aspartate Glutamate
Oxaloacetate -Ketoglutarate
Pyridoxamine phosphate
Pyridoxal phosphate
Glutamate oxaloacetate
transaminase (GOT)
Pyridoxal phosphate (PLP) first accepts the
amino group from aspartate
PLP is converted into pyridoxamine
phosphate and aspartate into oxaloacetate
Pyridoxamine phosphate then transfers the
amino group to -ketoglutarate
-Ketoglutarate is converted into glutamate
and pyridoxamine phosphate into PLP
An amino acid is converted into -keto
acid
A different -keto acid is converted into an
amino acid
Transamination is a coupled reaction
Though pyridoxal phosphate is a reactant,
the reaction is usually shown as:
The coenzyme goes back to its original
form at the end of the reaction
Aspartate +-Ketoglutarate GOT Oxaloacetate +Glutamate
PLP
Sometimes, the change in the coenzyme is
more important than that in the substrate
In glycolysis, glucose is oxidized to pyruvate,
and NAD+ is reduced in one reaction
Reduced NAD+ transfers its hydrogen atoms
to oxygen, and NAD+ is regenerated
In anaerobic conditions, NAD+ cannot be
regenerated due to lack of oxygen
One more reaction occurs in which pyruvate
is reduced to lactate
NADH is oxidized to NAD+ in this reaction
Here, regeneration of NAD+ is more
important for continuation of glycolysis
Glucose
↓
1,3-Biphosphoglycerate
↓
↓
↓
↓
↓
Glyceraldehyde-3-P
NAD+ NADH+H+
Lactate (Anaerobic) Pyruvate (Aerobic)
Enzyme nomenclature and classification
The nomenclature of enzymes has
undergone many changes over the years
The names given to enzymes in the
beginning were vague and uninformative
Some of the early names are pepsin,
ptylin, zymase etc
These names give no information about
the reaction catalyzed by the enzyme
Later on, a slightly more informative
nomenclature was adopted
Suffix -ase was added to the name of
the substrate e.g. lipase, protease etc
Still the type of reaction catalyzed 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 dehydro-
genase, pyruvate carboxylase, glutamate
decarboxylase etc
Even these names do not give complete
information, for example whether a coenzyme
is required or a byproduct is formed
International Union of Biochemistry (IUB) formed
an Enzyme Commission to make the names of
enzymes informative and unambiguous
The enzyme commission proposed a method of
nomenclature and classification of enzymes
which is applicable to all living organisms
According to IUB system:
• The enzymes have been divided into six
classes (numbered 1 - 6)
• Each class is divided into subclasses
• Subclasses are divided into subsub-
classes
• Subsubclasses are divided into
individual enzymes
The name of the enzyme has two parts
First part includes the name(s) of the sub-
strate(s) including cosubstrate (coenzyme)
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
IUB nomenclature
For example, the enzyme having the
trivial name glutamate dehydrogenase
catalyzes the following reaction:
L-Glutamate +NAD(P)+ +H2 O →
-Ketoglutarate +NAD(P)H +H+ +NH3
The IUB name of this enzyme is
L-Glutamate: NAD(P) oxidoreductase
(deaminating)
The IUB name shows that:
This enzyme acts on L-glutamate
NAD+ or NADP+ is required as a co-substrate
Type of reaction is oxido-reduction i.e. L-glutamate
is oxidised and the co-substrate is reduced
The amino group of L-glutamate is released as
ammonia
Moreover, each enzyme has been given
a code number consisting of four digits:
First digit ‒ Number of the class
Second digit ‒ Number of the subclass
Third digit ‒ Number of subsubclass
Fourth digit ‒ Number of the enzyme
The code number of L-glutamate: NAD(P)
oxidoreductase (deaminating) is EC 1.4.1.3
This shows that is it the third enzyme of
subsubclass 1 of subclass 4 of class 1
EC is the acronym for Enzyme Commission
The enzymes are
divided into six
classes in IUB
classification:
Oxidoreductases
Transferases
Hydrolases
Lyases
Isomerases
Ligases
Oxidoreductases
These are the enzymes that catalyze
oxidation-reduction reactions
One of the substrates is oxidised
and the other is reduced
Different subclasses act on
different chemical groups
Groups undergoing the reaction include
–CH=CH–, >CH–OH, >C=O, >CH–NH2 etc
Examples of oxidoreductases are:
Glutamate dehydrogenase
Lactate dehydrogenase
Malate dehydrogenase
Glycerol-3-phosphate dehydrogenase
Transferases
Transferases transfer a group other
than hydrogen from one substrate to
another
Such groups include methyl group,
amino group, phosphate group, acyl
group, glycosyl group etc
Examples of transferases are:
Hexokinase
Glucokinase
Glutamate oxaloacetate
transaminase
Ornithine carbamoyl transferase
Hydrolases
Hydrolases hydrolyse bonds such as
peptide, ester, glycosidic bonds etc
They are commonly found in the
digestive secretions and lysosomes
They hydrolyse carbohydrates, lipids,
proteins etc
Examples of
hydrolases are:
Amylase
Lipase
Pepsin
Ribonuclease
Lyases
Lyases remove chemical groups
from substrates by mechanisms
other than hydrolysis
The groups removed may be water,
amino group, carboxyl group etc
Examples of
lyases are:
Aldolase
Enolase
Fumarase
Isomerases
Isomerases catalyse inter-conversion
of isomers of a compound
Substrates include aldose-ketose
isomers, stereo-isomers etc
Examples of isomerases are:
Alanine racemase
Triose phosphate isomerase
Phosphohexose isomerase
Ribose-5-phosphate
ketoisomerase
Ligases
These enzymes ligate or bind two
substrates together
Binding occurs by a covalent bond
A source of energy is required e.g.
a high-energy phosphate
Examples of ligases are:
Glutamine synthetase
Squalene synthetase
Acetyl CoA carboxylase
Mechanism of action of enzymes
At temperatures above absolute zero i.e.
‒273°C, molecules are in constant motion
This movement is because of the kinetic
energy of the molecules
↑
← ●→
↓
A reaction occurs when reactant
molecules collide with each other (kinetic
theory of reaction)
But the reactant molecules must be in the
correct orientation when they collide
Correct
orientation
Incorrect
orientation
Energy input required to reach the critical
level is known as the energy of activation
Energy level of reactants has to be above
a critical level for the reaction to occur
The rate of reaction depends upon the
frequency of collisions between the
reactant molecules
The frequency of collisions can be
increased by raising the temperature
A rise in temperature
would increase:
Molecular motion
Frequency of collisions
Rate of reaction
The option of raising temperature is not
available in living organisms
In living organisms, the enzymes provide
an alternate pathway for the reaction
Enzymes lower the energy of activation
Enzymes.pptx
Enzymes.pptx
Enzymes.pptx
Enzyme-substrate interaction
The enzyme molecules are much larger
than their substrates
An enzyme possesses a specific binding
site for its substrate(s)
This site is known as the substrate site
(active site) of the enzyme
The substrate binds to the substrate site
forming enzyme-substrate (ES) complex
The binding may bring two substrates in
close proximity (bond-forming distance)
in the correct orientation so that a bond
is formed between
the two
The binding of a substrate to the enzyme
may induce a strain in the substrate
As a result, a bond is broken in the
substrate
The substrate is split into two or more
products which are released
Substrate ‒
Enzyme ‒
Products ‒
Substrate binds
to enzyme
A strain occurs in the
substrate; a bond is
broken
Substrate splits into
products which are
released
On binding of two substrates to the
enzyme, a chemical group may be
transferred from one substrate to
another
The catalytic action of the
enzyme may be exerted by:
Cofactors
Coenzymes
Some amino acid residues in
the substrate site
In the reaction catalysed by carbonic
anhydrase, the cofactor (zinc) catalyses
the reaction
– Zn++
H+ +HCO3
‒
H2O
H
– Zn++...‒O +H+
I
CO2
– Zn++ ‒ +
H
I
+O‒C‒O +H
II
O
– Zn++...O‒C‒O
II
O
H H
I I
+
H
I
– Zn ...O +C =O...H
II
O
++ ‒
In transamination reactions, the coenzyme
is involved in catalysis
The coenzyme (pyridoxal phosphate) is
present at the substrate site
It accepts an amino group from an amino
acid, and then donates it to a keto acid
Enzymes.pptx
Coenzyme
Amino acids participating in catalysis are
serine, histidine, cysteine, aspartate etc
In serine proteases, a serine residue at
the active site catalyses proteolysis
Examples of serine proteases are trypsin,
chymotrypsin, thrombin etc
Enzymes.pptx
The first model
was proposed by
Emil Fischer
Also known
as rigid
template
model
A different model
was later
proposed by
Koshland
Also known
as induced
fit model
Models of enzyme conformation
Fischer’s
model
Conformation of enzymes
very rigid
Lock and key type of
complementarity between
substrate and enzyme
Complementarity
responsible for specificity
of enzymes
Lock
Key
Fischer’s model
Fischer’s model did not agree with
certain experimental findings obtained
later
Conformation of enzyme was found to
change when it combined with its
substrate
Before substrate
binding
Enzyme
After substrate
binding
Substrate
Koshland’s model conforms to known findings
In the absence of substrate,
complementarity between enzyme and
substrate is not apparent
Approach of substrate induces change in
conformation of the enzyme
The substrate site becomes
complementary to the substrate
The substrate binds to the enzyme, and is
converted into the product
Release of the product restores the
enzyme to its original conformation
Change in conformation of the enzyme
produces ‘induced fit’
Koshland’s model
Allosteric enzymes
Some enzymes possess a site in
addition to the substrate site
This site is known as the allosteric
site
Such enzymes are known as
allosteric enzymes
Allosteric site is meant for binding of an
allosteric molecule
Binding of allosteric molecule changes the
conformation of substrate site
The allosteric molecule
is also known as:
Allosteric effector
Allosteric modifier
Allosteric regulator
Some allosteric molecules:
Facilitate the conformational
change required for substrate binding
They are known as allosteric
activators (positive modifiers)
They activate the enzyme
Enzyme
Substrate site
Allosteric site
Substrate
Allosteric
activator
Allosteric activator
binds to enzyme;
substrate site
changes
Substrate can
now bind to
substrate site
N-Acetylglutamate is an example
of allosteric activator
It activates carbamoyl phosphate
synthetase
Carbamoyl phosphate + 2 ADP + Pi
CO2 +NH3 +2 ATP
Carbamoyl
phosphate
synthetase
N-Acetylglutamate
⊕
Some allosteric regulators:
Prevent the conformational change
required for the binding of the substrate
Such regulators are known as allosteric
inhibitors (negative modifiers)
An example is glucose-6-phosphate
which inhibits hexokinase
Enzymes.pptx
Allosteric enzymes are usually present
at the start of long pathways
The allosteric inhibitor is generally the
product of the pathway
The allosteric enzyme regulates the
rate of formation of the product
In case the product is not being utilized,
it will accumulate
It will inhibit the allosteric enzyme; further
synthesis of the product will cease
When the product is used up, the
enzyme becomes free and active again
E1 is an
allosteric enzyme, and
P is its allosteric inhibitor
S I1 I2 I3 I4 P
E1 E2 E3 E4 E5
‒
Factors affecting the rates of
enzyme-catalysed reactions
Enzyme concentration
Substrate concentration
Coenzyme concentration
Temperature
pH
Enzyme concentration
First step in an enzyme-catalysed reaction
is formation of enzyme-substrate complex
The enzyme-substrate complex dissociates
into the enzyme and the product
It is regenerated in its original form at the
end of the reaction
E +S ↔ E S ↔ E +P
The enzyme may be considered to take
part in the reaction
Rate of the first reaction (formation of ES)
is proportional to the product of molar
concentrations of E and S
Rate of formation of ES  [E] [S]
Rate of the second reaction (formation of
E and P) is proportional to molar
concentration of ES
Rate of formation of E and P  [ES]
Therefore, the rate of the overall reaction
is proportional to the enzyme
concentration
But this is true only if enough substrate is
available to combine with the enzyme
Rate of reaction should be proportional
to substrate concentration also
But this is possible only if enough
enzyme is available to bind the substrate
However, the availability of enzymes in
the cells is limited
Substrate concentration
When the substrate concentration rises,
initially the velocity of the reaction rises
proportionately
But later the rise in velocity becomes less
until a maximum velocity (Vmax) is reached
Vmax
v
[S]
Vmax
2
Km
Plot between substrate concentration
and velocity
At Vmax, all the enzyme molecules are
saturated with the substrate
The velocity cannot increase further if the
substrate concentration is raised
The substrate concentration at which the
velocity is half of Vmax is known as
Michaelis constant (Km) of the enzyme
Vmax.[S]
v =
Km +[S]
The relationship between velocity of
reaction and the substrate concentration is
given by Michaelis-Menten
equation
v =
Vmax. [S]
Since both Vmax and Km are constant,
v  [S]
When the substrate concentration is very
low, the sum of Km and [S] is nearly
equal to Km as [S] is negligible
Hence, the equation may be rewritten as:
Km Km
v =
Vmax x [S]
or
[S] and [S] are cancelled;
the equation may be rewritten as:
v =Vmax
Vmax.[S]
v =
[S]
When the substrate concentration is very
high, the sum of Km and [S] is nearly
equal to [S] as Km is relatively negligible
Hence, the equation may be rewritten as:
Thus, when the substrate concentration
is equal to Km, the velocity is half of Vmax
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:
Vmax. [S] Vmax
v = =
2 [S] 2
Determination of Km
Every enzyme has got a
characteristic Km
Determination of Km is important in:
Study of
enzyme
kinetics
Assay of
enzyme
activity
Evaluation
of enzyme
inhibitors
Plotting v versus [S] is a lengthy process
Velocity has to be measured at a number
of substrate concentrations
The substrate concentration has to be
raised until Vmax is reached
Lineweaver and Burk devised a simple
method for determination of Km
Velocity is measured at a small number
(5-6) of substrate concentrations
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
Double
reciprocal plot
v =
Vmax.[S]
Km +[S]
Michaelis-Menten equation
1 =
Km +[S]
or
v [S]
Vmax Vmax
1 Km 1 1
=  +
Michaelis-Menten equation is
inverted
or
v Vmax.[S]
Km
1 =
v Vmax.[S]
[S]
+
Vmax.[S]
This is the equation for a straight line
y (y-axis) is 1/v
a (slope of the line) is Km/Vmax
x (x-axis) is 1/[S]
b (y-intercept) is 1/Vmax
1 = Km  1 1
+
v Vmax
y = a
[S] Vmax
x + b
At the x-intercept (where the line meets
the x-axis), the value of y =0
Therefore, at the x-intercept:
ax + b = 0
or ax = –
or x = –
b
b
a
or
On substituting the values of b and a:
x =
1

Km
Vmax Vmax
1
Vmax
or x = 
Km
Vmax
x =
Km
 1
Thus, the value of 1/[S] at the x-intercept
is 1/Km, and its reciprocal will be the Km
1
[S]
1
Km
1
v
1
Vmax
Allosteric enzymes do not follow
Michaelis-Menten equation
The v versus [S] plot of allosteric
enzymes is sigmoidal
This shows co-operative binding of
substrate to the enzyme
[S] → [S] →
↑
v
↑
v
Substrate concentration vs velocity plot
Normal
enzyme
Allosteric
enzyme
↑
v
[S] →
Positive effectors shift the plot to the left,
and negative effectors shift it to the right
Effect of allosteric activator and inhibitor on velocity
Kinetics of allosteric enzymes follow the
Hill equation
Hill plot is plotted between log v/Vmax–v
and log [S]
S50 of allosteric enzymes can be
determined from the Hill plot
S50 is the substrate concentration at
which the velocity is half of Vmax
In coenzyme-requiring reaction, coenzyme
concentration of also affects the velocity
Some coenzymes form an integral part of
the holoenzyme molecule
Other coenzymes act as co-substrates in
the reaction
Coenzyme concentration
If coenzyme is an integral part of enzyme,
the effect of coenzyme concentration is
same as that of enzyme concentration
If coenzyme acts as a second substrate,
the effect of coenzyme concentration is
similar to that of substrate concentration
To see the effect of temperature, velocity
of the reaction is measured at different
temperatures
A curve is plotted between velocity and
temperature
A bell-shaped curve is obtained
Temperature
↑
v
Optimum
temp
│
Temp →
Effect of temperature on velocity
When the temperature rises, the velocity
initially increases
This is due to increased kinetic energy of
the reactants
A further rise in temperature leads to
progressive denaturation of the enzyme
The velocity begins to decrease as the
enzyme gets denatured
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
The optimum temperature for all human
enzymes is 37°C
The temperature coefficient (Q10) of an
enzyme is the number of times the velocity
rises when temperature rises by 10°C
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
A bell-shaped curve is obtained
To see
the
effect of
pH:
Velocity is
measured at
different pH
levels
Velocity is
plotted
against pH
Optimum
pH
│
↑
v
pH →
Effect of pH on velocity
A change in pH alters electrical charges
on the enzyme molecules, and often on
substrate molecules as well
This can affect 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 because:
The electrical charges on the enzyme and
the substrate are the most suitable for:
Enzyme-substrate
binding
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
Enzyme inhibition
Catalytic activity of enzymes can be
inhibited by some compounds
Enzyme inhibition may be of two
types:
Competitive Non-competitive
Competitive inhibition
Competitive inhibition is also known as
substrate-analogue inhibition
The inhibitor has a close structural
resemblance with the substrate
Inhibitor can also bind to the substrate site of
enzyme because of structural resemblance
When inhibitor (I) binds to the enzyme,
enzyme-inhibitor (EI) complex is formed
However, the inhibitor cannot form the
product
Thus, in the presence of the inhibitor,
catalytic activity of the enzyme is inhibited
Substrate
Inhibitor
Products ‒‒
The inhibitor competes with the substrate
to bind to the enzyme
Hence, this type of inhibition is known as
competitive inhibition
Substrate
+
Inhibitor
+
Enzyme
Inhibitor
+
Enzyme
Substrate
+
Enzyme
I I
S
E
E E
When several molecules of substrate,
inhibitor and enzyme are present together:
Some enzyme
molecules bind the
substrate forming
ES complex
Some enzyme
molecules bind the
inhibitor forming
EI complex
Both ES and EI complexes are formed but
only ES can form the product
E +P
E
S I
No P
▼
ES ◀ ► EI
▼
Amounts of ES and EI complexes depend
upon the relative concentrations of S and I
If concentration of I
is higher
Less product will be
formed
More EI complex
will be formed
If concentration of
S is higher
Inhibition of
enzyme will be less
More ES complex
will be formed
If a Lineweaver-Burk plot is plotted in the
presence of a competitive inhibitor:
The y-intercept
remains unchanged
The x-intercept is
changed
1/[S] →
1 1
Km K’m
1
Vmax
– In the presence
of inhibitor
↑
1
v
Competitive inhibition
– In the absence
of inhibitor
The y-intercept is 1/Vmax which remains
unchanged in the presence of competitive
inhibitor
The x-intercept is 1/Km which becomes
less in the presence of competitive
inhibitor
Competitive inhibitors do not affect the
Vmax
The Vmax can be attained even in the
presence of the inhibitor
But more substrate is required to reach
the Vmax in the presence of the inhibitor
Efficacy of a competitive inhibitor can be
assessed by measuring Km:
The extent of rise in Km is a measure of
efficacy of the inhibitor
In the presence
of the inhibitor
In the absence
of the inhibitor
Competitive inhibitors of some enzymes
are being used as drugs
They are used to inhibit specific reactions
The inhibition produces a desired
pharmacological effect
Some competitive inhibitors
used as drugs are:
Amethopterin and aminopterin
Allopurinol
Physostigmine and neostigmine
Mevastatin and lovastatin
Amethopterin and aminopterin
Are structural analogues of folic acid
Inhibit dihydrofolate reductase
H2N N
1
2
N 3
4
|
OH
5
6
N
7
N
8
9 10
CH2— N —
|
H
— C — N — CH
|
COOH
COOH
|
CH2
|
O H CH2
|| | |
CH3
Folic acid
Amethopterin
H2N N
1
2
N 3
4
|
OH
5
6
N
7
N
8
9 10
CH2—N — — C — N — CH
|
COOH
COOH
|
CH 2
|
O H CH2
|| | |
|
CH3
Dihydrofolate
reductase
Folate
Dihydrofolate
reductase
Tetrahydrofolate
Tetrahydrofolate is required for the synthesis
of purine and thymine nucleotides
NADPH +H+
NADPH+
Dihydrofolate
NADPH +H+
NADPH+
Inhibition of dihydrofolate reductase
decreases the synthesis of nucleotides
Decreased availability of nucleotides
decreases DNA synthesis and cell division
Thus, cell division is suppressed in the
presence of amethopterin and aminopterin
Therefore, they are used as anti-cancer
drugs
Allopurinol
Is a structural analogue
of hypoxanthine
It inhibits xanthine
oxidase
N
HN
O
||
C
C
C
HC
N
H
C
N
H
Allopurinol
HN
O
||
C
C
C
HC
N N
H
N
CH
Hypoxanthine
Hypoxanthine
Xanthine
oxidase
Xanthine
Xanthine
oxidase
Uric acid
Xanthine oxidase converts hypoxanthine
into xanthine
Then, it converts xanthine into uric acid
Allopurinol is used to treat gout
Gout results from over-production of uric
acid
Allopurinol inhibits the formation of uric
acid
Physostigmine and neostigmine
Are structural analogues of
acetylcholine
They inhibit acetyl
cholinesterase
Acetylcholine
Choline
Acetyl
cholinesterase
H2O
Acetate ◀
▼
Physostigmine and neostigmine decrease
the breakdown of acetylcholine
They are used to treat myasthenia gravis,
an auto-immune disorder
Number of acetylcholine receptors is
decreased in myasthenia gravis
Mevastatin and Lovastatin
Are structural analogues of
HMG CoA
Are inhibitors of HMG CoA
reductase
Mevalonate
HMG CoA
HMG CoA
reductase
Cholesterol
Therefore, mevastatin and lovastatin are
used as hypo-cholesterolaemic drugs
Inhibition of this reaction decreases the
synthesis of cholesterol
HMG CoA reductase catalyses the key
reaction in the synthesis of cholesterol
Non-competitive inhibition
The non-competitive inhibitors have no
structural resemblance with the substrate
They do not compete with the substrate for
binding to the enzyme
They bind to some other region of the
enzyme and render it inactive
Enzyme +Substrate
+ Inhibitor
Enzyme +Substrate
Non-competitive inhibition
Non-competitive inhibition may be
reversible or irreversible
Generally, it is irreversible
Examples are iodoacetamide, cyanide, p-
chloromercuribenzoate, heavy metals etc
If a Lineweaver-Burk plot is plotted in the
presence of a non-competitive inhibitor:
The y-intercept
becomes higher
The x-intercept
remains unchanged
In the presence
of inhibitor
In the absence
of inhibitor
↑
1
v
1/[S] →
1
Km
1
Vmax
1
V’max
Non-competitive inhibition
Non-competitive inhibitors decrease the
Vmax but do not affect the Km
The substrate concentration required to
reach the new Vmax remains unchanged
Chemical reactions in living organism are
usually parts of some metabolic pathway
A pathway consists of a series of
reactions
Each pathway serves some specific
purpose(s)
Regulation of enzymes
Metabolic pathways need to be regulated
precisely
Regulation ensures adequacy of products
with no wastage of raw materials
Requirements of the organism keep on
changing
Regulatory mechanisms must be
responsive to these changes
Concentrations of enzymes
Enzymes play a crucial role in
the regulatory mechanisms
Metabolic pathways are regulated
by changing one of the following:
Catalytic activity of enzymes
Rate-limiting step in the pathway
Committed step in the pathway
The regulation involves one or a few
“key” enzymes in a pathway
The key enzyme (or regulatory enzyme)
may catalyse:
Rate-limiting
step
An early reaction
that controls the
availability of
substrates for the
subsequent
reactions
Committed
step
The earliest
irreversible
reaction unique
to the pathway
Regulation of enzyme concentration
Some pathways are regulated by altering
the concentrations of the key enzyme(s)
If the enzyme concentration increases, the
rate of reactions would increase
If the enzyme concentration decreases, the
rate of reactions would decrease
Enzyme concentration can be altered by
increasing or decreasing:
Rate of synthesis
of enzyme
Regulation of enzyme synthesis is
commoner
Rate of breakdown
of enzyme
Regulation of enzyme synthesis
Enzyme synthesis may be
regulated by:
Induction of enzyme synthesis
Repression of enzyme synthesis
Conversion of proenzyme into
enzyme
Constitutive
enzymes
Inducible
enzymes
Induction
Enzymes may be divided into:
Constitutive
enzymes
Inducible
enzymes
Continuously
synthesized
Synthesized only
when required
Always present in
the cell
Synthesized when
inducer enters the
cell
Inducer may be the substrate for the
enzyme or may be a gratuitous inducer
A gratuitous inducer is one which is
not a substrate for the enzyme
Inducer acts on DNA; increases expression
of the gene encoding the enzyme
An example is induction of key enzymes
of gluconeogenesis by glucocorticoids
Synthesis of some enzymes is regulated
by repression
Transcription of gene encoding the
enzyme is blocked by a repressor
The repressor is made up of apo-
repressor and co-repressor
Repression
X
X
Repression of gene expression
Apo-repressor is a protein always
present in the cell
When co-repressor enters or
accumulates, it combines with apo-
repressor to form the repressor
The co-repressor is generally the
product of the pathway
An example is regulation of haem
synthesis
The regulatory enzyme is -aminolevulinic
acid synthetase
Haem is the regulator of this enzyme
Haem acts as co-repressor; combines
with aporepressor to form repressor
The repressor represses the synthesis of
this early enzyme in the pathway
Decreased enzyme availability decreases
haem synthesis
When haem is used up, the repressor
cannot be formed
The repression is relieved; the enzyme
synthesis re-commences
This is known as derepression
Conversion of proenzyme into enzyme
Sometimes, the concentration of enzymes
needs to be increased quickly
For example, when food enters stomach,
pepsin concentration has to be raised quickly
This cannot be done by induction or
derepression which are slow processes
The enzyme is synthesized in the form of
a precursor, pepsinogen
Pepsinogen is an inactive proenzyme
The proenzyme will not digest the mucosal
proteins
Entry of food in the stomach generates
some signals
These signals convert pepsinogen into
pepsin
The enzyme concentration is raised
quickly
Proenzyme
Peptide
Proteolysis
Active site
(masked) →
Active site
(exposed) →
Enzyme
Proteolytic activation of proenzyme
Substrate
Regulation of enzyme degradation
Enzyme concentration may also be
regulated by altering its breakdown
Increased breakdown will decrease the
concentration of the enzyme
Decreased breakdown will increase the
concentration of the enzyme
Regulation of degradation is not common
in higher organisms
A few examples are seen in starvation in
which nutrients need to be conserved
Concentration of some enzymes is
increased by decreasing their breakdown
An example is tryptophan pyrrolase
Regulation of catalytic activity
of enzymes
The key enzyme is regulated by altering its
catalytic activity
Enzyme concentration remains unchanged;
catalytic activity is increased or decreased
Catalytic activity of the enzyme may be
altered by:
Allosteric
regulation
of the enzyme
Covalent
modification
of the enzyme
Allosteric regulation
This mechanism is used in some long
metabolic pathways
The substrate is converted into a product
by a series of reactions
The earliest functionally irreversible reaction
is catalysed by an allosteric enzyme
Usually, the product of the pathway is
the allosteric inhibitor of the enzyme
When the product accumulates, it
inhibits the allosteric enzyme
S I1 I2 I3 I4 P
E1 E2 E3 E4 E5
‒
When the product is used up, the inhibition
is relieved
Thus, synthesis of the product is regulated
according to rate of its utilization
If there are a number of irreversible steps,
regulation may occur at a number of steps
Some enzymes are regulated by positive
allosteric modulation (i.e. activation)
An example is the first reaction of urea
cycle
This reaction is catalyzed by carbamoyl
phosphate synthetase I (mitochondrial)
Carbamoyl phosphate + 2 ADP + Pi
N-Acetylglutamate
⊕
Carbamoyl phosphate synthetase I is an
allosteric enzyme
It is allosterically activated by N-acetyl-
glutamate
CO2 +NH3 +2 ATP
Carbamoyl
phosphate
synthetase I
Many enzymes are regulated by negative
allosteric modulation (i.e. inhibition)
An example is asparate transcarbamoylase
It is an early enzyme in de novo synthesis
of pyrimidine nucleotides
It is inhibited by cytidine triphosphate, a
product of the pathway
A few enzymes are subject to positive as
well as negative allosteric regulation
Phosphofructokinase-1, a regulatory
enzyme in glycolytic pathway, is subject to:
Allosteric activation
by AMP
Allosteric inhibition
by ATP
The enzymes regulated by this mechanism
can exist in two forms
The two forms can be converted into each
other
The conversion occurs by a covalent
modification of the enzyme molecule
Covalent modification
During conversion, a covalent bond is
either formed or broken in the enzyme
The most common covalent modification is
addition or removal of phosphate
Phosphate is usually added to or removed
from a serine residue in the enzyme
The phosphate group is added by a
protein kinase
It is removed by a protein phosphatase
Protein kinase
Protein
phosphatase
H2O
ATP
Pi
Enzyme‒Ser‒OH
(Dephosphorylated
enzyme)
Enzyme‒Ser‒O‒ P
(Phosphorylated
enzyme)
► ADP
Out of the two forms of the enzyme, one is
active and the other inactive
The form depends upon relative activities of
protein kinase and protein phosphatase
These, in turn, are controlled by hormones
acting through second messengers
An example is glycogen synthetase, the
regulatory enzyme of glycogenesis
Its dephosphorylated form is active and the
phosphorylated form is inactive
Protein
kinase A
Protein
phosphatase-1
H2O
ATP
Pi
Glycogen
synthetase
(Active)
Glycogen
synthetase‒ P
(Inactive)
► ADP
Another example is glycogen phospho-
rylase, the key enzyme of glycogenolysis
Its phosphorylated form is active and the
dephosphorylated form is inactive
Phosphorylase
kinase a
Protein
phosphatase-1
H2O
ATP
Pi
Phosphorylase
(Inactive)
Phosphorylase‒ P
(Active)
► ADP
Some enzymes are regulated by
multiple mechanisms
For example, acetyl CoA carboxylase
is subject to:
Induction
Repression
Allosteric regulation
Covalent modification
A large number of enzymes are
synthesized in various cells
They are continuously released into
circulation due to natural cell death
They are continually removed from
circulation by degradation or excretion
Enzymes of diagnostic importance
The circulating enzymes may be divided
into two types:
Functional plasma enzymes or
plasma-specific enzymes
Non-functional plasma enzymes
or non-plasma-specific enzymes
The enzymes are normally present in
circulation in very low concentrations
Functional plasma enzymes
These enzymes are purposely secreted
into circulation
They perform specific catalytic functions in
plasma
Examples are lipoprotein lipase, blood
clotting factors, complement proteins etc
Non-functional plasma enzymes
These enzymes do not perform any
function.in plasma
These are intracellular enzymes which
enter the circulation when the cells in
which they are synthesized die
Non-functional plasma enzymes or non-
plasma-specific enzymes
These enzymes do not perform any
function.in plasma
These are intracellular enzymes which
enter the circulation when the cells in
which they are synthesized die
When cell death is occurring at normal
rate, non-functional enzymes are released
in very small amounts
Their concentrations in plasma remain very
low
If the rate of cell death increases, these
enzymes are released in large amounts
Their concentrations in plasma can rise
many times above normal
A non-functional plasma enzyme can pin-
point the site of the disease
IF
It has a selective tissue distribution
OR
If its concentration is far higher in some
tissues than elsewhere in the body
Thus, the enzymes of
diagnostic importance are:
The non-functional plasma
enzymes ‒
Having a selective tissue
distribution
Plasma enzymes that are established
diagnostic tools:
• Lactate dehydrogenase (LDH)
• Transaminases (GOT and GPT)
• Creatine kinase (CK)
• Gamma-glutamyl transpeptidase (GGT)
• Alkaline phosphatase (ALP)
• Acid phosphatase (ACP)
• Amylase
• Lipase
• Ceruloplasmin
Lactate
dehydrogenase
(LDH)
Catalyses
interconversion of
pyruvate and lactate
Tissue distribution
very wide
Concentration very
high in myocardium,
muscles and liver
Plasma LDH rises in:
Myocardial
infarction
Viral hepatitis
Muscle injuries
In myocardial infarction:
Rise begins 24 hours after
infarction
Peak value is reached in
about three days
Level returns to normal in
about a week
Transaminases
The two most important are GOT
and GPT
GOT is glutamate oxaloacetate
transaminase
GPT is glutamate pyruvate
transaminase
GOT is also known as aspartate
aminotransferase (AST)
GPT is also known as alanine
aminotransferase (ALT)
GOT and GPT are present
in high concentrations in:
Liver Muscles
Myocardium
Serum GOT and GPT
are raised in:
Myocardial infarction
Viral hepatitis
Muscle injuries
Concentration of GOT is higher than
that of GPT in myocardium while the
situation is reverse in liver
Therefore
Rise in plasma GOT is more in
myocardial infarction and that in
GPT is more in viral hepatitis
Creatine kinase (CK)
Also known as creatine
phosphokinase (CPK)
Catalyses interconversion of
creatine and creatine phosphate
Creatine +ATP ↔ Creatine~
℗+ADP
CK is present in:
Brain
Muscles Myocardium
Serum CK is raised in:
Myocardial infarction
Myopathies
Muscle injuries
Rise begins within 3-6 hours after MI
Peak is reached in 24 hours
Returns to normal in three days
Specific and early indicator of MI
Serum CK in myocardial infarction (MI)
Enzyme
level
Upper limit
of normal
0 1 2 3
Days
4 5 6 7
CK GOT LDH
Begins to
rise in
Reaches
peak in
Returns to
normal in
Specificity
Myoglobin 1-3 hrs 4-6 hrs 18-24 hrs Low
Cardiac
troponin T 4-6 hrs 18-36 hrs 5-15 days High
Cardiac
troponin I 4-6 hrs 12-24 hrs 5-10 days High
Non-enzyme markers of myocardial
infarction
Gamma-glutamyl
transpeptidase (GGT)
Transfers the -glutamyl residue
of glutathione to other substrates
Serum level increases in most of
the liver diseases
Is an early indicator of
alcoholic hepatitis
Alkaline phosphatase (ALP)
ALP is a group of enzymes
The group hydrolyses organic
phosphate esters
Its optimum pH is in alkaline range
ALP is released in circulation mainly
from bones and liver
Smaller amounts are released from
intestines and placenta
Liver excretes ALP in bile
A marked rise in plasma ALP
occurs in obstructive jaundice
Smaller elevations occur in:
Viral hepatitis
Rickets
Hyperparathyroidism
Osteosarcoma
Bony metastases
Acid phosphatase (ACP)
ACP is a group of enzymes
The group hydrolyses organic
phosphate esters
Its optimum pH is in acidic range
The main source of circulating ACP is
the prostate gland
Serum ACP is elevated in metastatic
carcinoma of prostate
Amylase
A digestive enzyme synthesized in
the pancreas and the parotid gland
Sharp elevation of serum amylase
occurs in acute pancreatitis
A smaller elevation occurs in
acute parotitis
Lipase
A lipolytic enzyme released into
circulation from the pancreas
Serum lipase rises in acute
pancreatitis
Ceruloplasmin
A copper-containing protein having
ferroxidase activity
Absent or very low in serum in Wilson’s
disease (hepatolenticular degeneration)
Isoenzymes
Multiple molecular forms of the same
enzyme
All catalyse the same reaction
They differ slightly in physical, chemical
and immunological properties
Isoenzymes possess quaternary
structure
They are made up of two or more sub-
units that are different from each other
The subunits have slightly different
primary structures
Isoenzymes usually differ in Km and Vmax
Their regulation may be different
This helps in fine-tuning of metabolism
Isoenzymes can be
separated by:
Electrophoresis
Chromatography
Immunochemical methods
The tissue distribution of isoenzymes
is highly specific
Measurement of isoenzymes can be of
great diagnostic importance
Isoenzymes of diagnostic
importance include:
Lactate dehydrogenase
Creatine kinase
Alkaline phosphatase
Lactate dehydrogenase
H subunit M subunit
First enzyme shown to exist in the form of
five isoenzymes by Markert (1957)
The enzyme is a tetramer made up of two
types of subunits – H and M
• HHHH
• HHHM
• HHMM
• HMMM
• MMMM
The subunits can form five different
tetramers (isoenzymes):
or LD1
or LD2
or LD3
or LD4
or LD5
or LDH1
or LDH2
or LDH3
or LDH4
or LDH5
The LD isoenzymes in plasma can be
separated by electrophoresis
The normal pattern of LD isoenzymes in
serum is LD2 >LD1 >LD3 >LD4 >LD5
The predominant isoenzymes in
myocardium are LD1 and LD2
Both are raised in myocardial infarction
The rise in LD1 is greater than that in LD2
Hence, the plasma LD isoenzyme pattern
becomes LD1 >LD2 >LD3 >LD4 >LD5
LD5 is the predominant isoenzyme in liver
Therefore, LD5 is raised in viral hepatitis
Creatine kinase
B subunit M subunit
A dimer made up of two types of
subunits
The subunits are – B and M
Three different dimers (isoenzymes) can
be formed from these two subunits:
• BB or CK1 or CK-BB
• MB or CK2 or CK-MB
• MM or CK3 or CK-MM
CK-MB is commonly measured by immuno-
inhibition
Serum is treated with anti-M subunit antibody
CK-MM is inhibited
The residual enzyme is taken to be CK-MB
as CK-BB is negligible
Normal CK isoenzyme pattern in plasma
CK-MB
CK-BB
CK-MM
The major isoenzyme in myocardium is
CK-MB
In plasma, CK-MB is less than 3% of
total CK
CK-MB is raised in myocardial infarction
CK-BB, CK-MB and CK-MM are present in
cytosol
A different CK is present in mitochondria –
mitochondrial CK (CK-MT or CK-Mi)
CK-MT has two isoforms: CK-MT1 and
CK-MT2
CK-MT1 is ubiquitous
CK-MT2 is present in skeletal and heart
muscle
CK-MT can exist as a dimer or an octamer
The dimeric and octameric forms are
inter-changeable
CK-MT1 and CK-MT2 are encoded by
different genes
Thus, there are four genes for CK
subunits
These are CK-M, CK-B, CK-MT1 and
CK-MT2 genes
CK-M and CK-B genes encode the
cytosolic enzyme
CK-MT1 and CK-MT2 genes encode the
mitochondrial enzyme
CK-MT1 and CK-MT2 isoenzymes have no
diagnostic importance
Bone, liver
, intestine and placenta form
different isoenzymes
ALP isoenzymes are commonly separated
by electrophoresis
Liver isoenzyme moves the fastest and
occupies the same position as 2-globulin
Alkaline phosphatase
The bone ALP closely follows the liver
ALP
The placental isoenzyme follows the bone
isoenzyme
The intestinal isoenzyme is the slowest
moving
The liver ALP is raised in liver cancer and
biliary obstruction
The bone ALP is raised in bone cancers
and Paget’s disease
The placental and intestinal isoenzymes
have no diagnostic importance
Two atypical ALP isoenzymes are seen
in some cancers
These are Regan isoenzyme and Nagao
isoenzyme
Regan and Nagao isoenzymes resemble
the placental isoenzyme
Regan isoenzyme is raised in cancer of
breast, lungs, colon, uterus and ovaries
Nagao isoenzyme is raised in germ cell
cancer of the testes
Assay of enzymes
Several enzymes present in circulation help
in diagnosis of diseases
For this, we need to measure serum levels
of these enzymes
Sometimes, such measurement is required
for academic purpose
Enzyme concentrations in serum are very
minute
Isolation and purification of enzymes is
difficult and time-consuming
Therefore, direct measurement of enzyme
concentrations is very difficult
Enzyme concentrations are measured
indirectly
Velocity of the enzyme-catalyzed reaction
is measured
Conditions are such that rate of reaction is
proportional to the enzyme concentration
The reaction is carried out in a fixed-
temperature water-bath or an incubator
Optimum pH is maintained by using a
buffer
Substrate concentration is kept constant
and high
Rate of reaction in such conditions is
proportional to the enzyme concentration
The rate of the reaction can be
determined by measuring:
The rate of disappearance
of the substrate
Rate of appearance of the
product
In endpoint methods:
The reaction is carried out for a fixed
period
Initial and final concentrations of the
substrate or the product are measured
In kinetic methods, the concentration of the
substrate or the product is measured at
regular intervals for a brief period
The result in both the methods is expressed
in arbitrary units of enzyme activity rather
than enzyme concentration
Many enzymes are used as tools in
diagnostic and research laboratories
Glucose oxidase and peroxidase are
used for measuring glucose concentration
Hexokinase and glucose-6-phosphate
dehydrogenase are also used for
measuring glucose concentration
Enzymes as laboratory tools
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
Peroxidase and alkaline phosphatase are
used to label antibodies in ELISA
A number of enzymes are used in
recombinant DNA technology e.g.
DNA ligase
Terminal transferase
S1 nuclease
Reverse transcriptase
Taq polymerase
Restriction endonucleases
Some human, animal, plant and microbial
enzymes are used as drugs also
Diastase, papain, pepsin, chymotrypsin etc
are used to aid digestion
Amylase, lipase and proteases are used in
the treatment of pancreatic insufficiency
Enzymes as drugs
Serratiopeptidase is a bacterial proteolytic
enzyme
It is used to remove dead tissue from the
site of inflammation to accelerate healing
It is also used to reduce inflammation,
oedema and pain
catalyses hydrolysis of
Hyaluronidase
hyaluronic acid
Hyaluronidase injections are used to
facilitate delivery of other injectable drugs
Asparaginase is used in the chemotherapy
of leukaemia
Leukaemic cells are deficient in asparagine
synthetase
For their asparagine requirement, they are
dependent on pre-formed asparagine
converts asparagine into
Asparaginase
aspartate
This deprives the leukaemic cells of an
essential nutrient
Thrombolytic drugs used to clear
blockage of blood vessels are:
Urokinase
Streptokinase
Tissue plasminogen activator
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Enzymes.pptx

  • 1. Enzymes The biological catalysts Dr. Sangeeta Khyalia M.D. (Biochemistry) Jaipur, India
  • 2. A vast multitude of chemical reactions occur in living organisms It is these reactions that keep the organism going These reactions would occur at extremely low velocities in the absence of catalysts
  • 3. Common catalysts used in non-living systems are: Acids Alkalis Metals These are not suitable for living organisms because of their: Toxicity Lack of specificity
  • 4. Biological catalysts should be: Safe (non-toxic) Specific (generally catalyzing only one reaction) Capable of adjusting their catalytic activity All these properties are present in enzymes
  • 5. Enzymes were first discovered in yeast (enzyme means ‘in yeast’) They were later found in other living organisms as well They could catalyze reactions outside the living organisms also Chemically, all enzymes were found to be proteins
  • 6. Definition Enzymes are protein catalysts that catalyse chemical reactions in biological systems But this definition is not entirely correct Some RNA molecules (ribozymes) have been found to catalyze some reactions
  • 7. The reactant on which the enzyme acts is known as the substrate of the enzyme The enzyme converts the substrate into a product or products Substrate Enzyme Product
  • 8. Enzyme specificity If an enzyme catalyses a number of reactions, it will be impossible to regulate individual reactions However, this doesn’t happen; the enzymes are highly specific
  • 9. Generally, one enzymes catalyses only one reaction This is of crucial importance for regulation of metabolic pathways However, the degree of specificity may differ in different enzymes
  • 10. Enzyme specificity may have the following orders: Group specificity Substrate specificity Stereo-specificity
  • 11. Group specificity Enzyme is specific for a chemical group or bond but not for the actual substrate Group-specific or bond-specific enzymes are commonly present in digestive secretions
  • 12. For example, pepsin is specific for peptide bond but not for any protein Thus, a large variety of dietary proteins can be digested by the same enzyme Trypsin, chymotrypsin, nucleases, lipases and glycosidases are other examples
  • 13. Some group-specific enzymes have a slightly higher degree of specificity For example, aminopeptidase hydrolyses only N-terminal peptide bond Carboxypeptidase hydrolyses only the C-terminal peptide bond Endopeptidases hydrolyse the internal peptide bonds only
  • 15. Most enzymes are specific for a chemical bond/group as well as the substrate For example, glucokinase and fructokinase are substrate-specific enzymes They transfer a phosphate group from ATP to one specific substrate Substrate specificity
  • 17. Stereo-specificity Many biomolecules exhibit stereo- isomerism Examples are carbohydrates and amino acids Enzymes acting on these are specific for one stereo-isomer
  • 18. Mammalian enzymes acting on carbo- hydrates are generally specific for D-isomers Those acting on amino acids are generally specific for L-isomers Exceptions are racemases which inter-convert the D- and L-isomers
  • 19. COOH I H2N – C – H COOH I H – C – NH2 Alanine racemase I CH3 L-Alanine I CH3 D-Alanine Stereospecificity – An exception
  • 20. Coenzymes and cofactors Some enzymes require a non-protein substance for their catalytic activity If the non-protein substance is organic, it is known as a coenzyme If the non-protein substance is inorganic, it is known as a cofactor
  • 21. In some cases, the coenzyme is an integral part of the enzyme In others, its presence is required during the reaction
  • 22. The protein portion of an enzyme that requires a coenzyme is called apoenzyme Apoenzyme +Coenzyme → Holoenzyme Apoenzyme combines with coenzyme to form the active holoenzyme
  • 24. The coenzymes generally contain vitamins of B-complex family Some act as coenzymes by them- selves e.g. biotin Others are converted into coenzymes
  • 25. B-Complex vitamins that are converted into coenzymes are: Thiamin Riboflavin Niacin Pantothenic acid Pyridoxine Folic acid Vitamin B12
  • 26. Coenzymes are generally required in group transfer reactions such as: Oxidation-reduction Transamination Phosphorylation
  • 27. Coenzymes can be divided into two groups: Coenzymes involved in transfer of hydrogen Coenzymes involved in transfer of groups other than hydrogen
  • 28. Coenzymes involved in transfer of hydrogen: Flavin mononucleotide (FMN) Flavin adenine dinucleotide (FAD) Nicotinamide adenine dinucleotide (NAD+) Nicotinamide adenine dinucleotide phosphate (NADP+) Lipoic acid Coenzyme Q
  • 29. Coenzymes involved in transfer of groups other than hydrogen: Thiamin pyrophosphate (TPP) Coenzyme A (Co A) Pyridoxal phosphate (PLP) Tetrahydrofolate (H4- Folate) Cobamides (B12- Coenzymes) Lipoic acid Biotin ATP & similar nucleotides
  • 30. Role of coenzymes The enzyme acts upon its substrate, and converts it into a product Coenzyme acts as a co-substrate (second substrate) in group transfer reactions The coenzyme donates or accepts the group that is being transferred
  • 31. EMB-RCG In the second reaction, the coenzyme NAD+ acts a second substrate and accepts the hydrogen atoms In the first reaction, the coenzyme ATP acts as a second substrate and donates a phosphate group CH2‒OH CH‒OH CH2‒OH CH‒OH CH2‒OH C =O CH2‒O‒ CH2‒OH ATP Glycerol Glycerol kinase ADP CH2‒O‒ Glycerol-3- phosphate Dihydroxy- acetone phosphate Glycerol-3- phosphate dehydrogenase NAD+ NADH +H+
  • 32. The chemical change in coenzyme is opposite to that in the substrate If the substrate loses a chemical group, the coenzymes accepts it If the substrate gains a chemical group, the coenzymes provides it
  • 33. They act only as carriers, and regain their original form at the end of the reaction Pyridoxal phosphate, for example, acts as a carrier of amino group in transamination Some coenzymes accept a group from one substrate and donate it to another
  • 34. Aspartate Glutamate Oxaloacetate -Ketoglutarate Pyridoxamine phosphate Pyridoxal phosphate Glutamate oxaloacetate transaminase (GOT)
  • 35. Pyridoxal phosphate (PLP) first accepts the amino group from aspartate PLP is converted into pyridoxamine phosphate and aspartate into oxaloacetate Pyridoxamine phosphate then transfers the amino group to -ketoglutarate -Ketoglutarate is converted into glutamate and pyridoxamine phosphate into PLP
  • 36. An amino acid is converted into -keto acid A different -keto acid is converted into an amino acid Transamination is a coupled reaction
  • 37. Though pyridoxal phosphate is a reactant, the reaction is usually shown as: The coenzyme goes back to its original form at the end of the reaction Aspartate +-Ketoglutarate GOT Oxaloacetate +Glutamate PLP
  • 38. Sometimes, the change in the coenzyme is more important than that in the substrate In glycolysis, glucose is oxidized to pyruvate, and NAD+ is reduced in one reaction Reduced NAD+ transfers its hydrogen atoms to oxygen, and NAD+ is regenerated
  • 39. In anaerobic conditions, NAD+ cannot be regenerated due to lack of oxygen One more reaction occurs in which pyruvate is reduced to lactate NADH is oxidized to NAD+ in this reaction Here, regeneration of NAD+ is more important for continuation of glycolysis
  • 41. Enzyme nomenclature and classification The nomenclature of enzymes has undergone many changes over the years The names given to enzymes in the beginning were vague and uninformative Some of the early names are pepsin, ptylin, zymase etc These names give no information about the reaction catalyzed by the enzyme
  • 42. Later on, a slightly more informative nomenclature was adopted Suffix -ase was added to the name of the substrate e.g. lipase, protease etc Still the type of reaction catalyzed by the enzyme remained unclear
  • 43. 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 dehydro- genase, pyruvate carboxylase, glutamate decarboxylase etc Even these names do not give complete information, for example whether a coenzyme is required or a byproduct is formed
  • 44. International Union of Biochemistry (IUB) formed an Enzyme Commission to make the names of enzymes informative and unambiguous The enzyme commission proposed a method of nomenclature and classification of enzymes which is applicable to all living organisms
  • 45. According to IUB system: • The enzymes have been divided into six classes (numbered 1 - 6) • Each class is divided into subclasses • Subclasses are divided into subsub- classes • Subsubclasses are divided into individual enzymes
  • 46. The name of the enzyme has two parts First part includes the name(s) of the sub- strate(s) including cosubstrate (coenzyme) 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 IUB nomenclature
  • 47. For example, the enzyme having the trivial name glutamate dehydrogenase catalyzes the following reaction: L-Glutamate +NAD(P)+ +H2 O → -Ketoglutarate +NAD(P)H +H+ +NH3 The IUB name of this enzyme is L-Glutamate: NAD(P) oxidoreductase (deaminating)
  • 48. The IUB name shows that: This enzyme acts on L-glutamate NAD+ or NADP+ is required as a co-substrate Type of reaction is oxido-reduction i.e. L-glutamate is oxidised and the co-substrate is reduced The amino group of L-glutamate is released as ammonia
  • 49. Moreover, each enzyme has been given a code number consisting of four digits: First digit ‒ Number of the class Second digit ‒ Number of the subclass Third digit ‒ Number of subsubclass Fourth digit ‒ Number of the enzyme
  • 50. The code number of L-glutamate: NAD(P) oxidoreductase (deaminating) is EC 1.4.1.3 This shows that is it the third enzyme of subsubclass 1 of subclass 4 of class 1 EC is the acronym for Enzyme Commission
  • 51. The enzymes are divided into six classes in IUB classification: Oxidoreductases Transferases Hydrolases Lyases Isomerases Ligases
  • 52. Oxidoreductases These are the enzymes that catalyze oxidation-reduction reactions One of the substrates is oxidised and the other is reduced Different subclasses act on different chemical groups Groups undergoing the reaction include –CH=CH–, >CH–OH, >C=O, >CH–NH2 etc
  • 53. Examples of oxidoreductases are: Glutamate dehydrogenase Lactate dehydrogenase Malate dehydrogenase Glycerol-3-phosphate dehydrogenase
  • 54. Transferases Transferases transfer a group other than hydrogen from one substrate to another Such groups include methyl group, amino group, phosphate group, acyl group, glycosyl group etc
  • 55. Examples of transferases are: Hexokinase Glucokinase Glutamate oxaloacetate transaminase Ornithine carbamoyl transferase
  • 56. Hydrolases Hydrolases hydrolyse bonds such as peptide, ester, glycosidic bonds etc They are commonly found in the digestive secretions and lysosomes They hydrolyse carbohydrates, lipids, proteins etc
  • 58. Lyases Lyases remove chemical groups from substrates by mechanisms other than hydrolysis The groups removed may be water, amino group, carboxyl group etc
  • 60. Isomerases Isomerases catalyse inter-conversion of isomers of a compound Substrates include aldose-ketose isomers, stereo-isomers etc
  • 61. Examples of isomerases are: Alanine racemase Triose phosphate isomerase Phosphohexose isomerase Ribose-5-phosphate ketoisomerase
  • 62. Ligases These enzymes ligate or bind two substrates together Binding occurs by a covalent bond A source of energy is required e.g. a high-energy phosphate
  • 63. Examples of ligases are: Glutamine synthetase Squalene synthetase Acetyl CoA carboxylase
  • 64. Mechanism of action of enzymes At temperatures above absolute zero i.e. ‒273°C, molecules are in constant motion This movement is because of the kinetic energy of the molecules ↑ ← ●→ ↓
  • 65. A reaction occurs when reactant molecules collide with each other (kinetic theory of reaction) But the reactant molecules must be in the correct orientation when they collide Correct orientation Incorrect orientation
  • 66. Energy input required to reach the critical level is known as the energy of activation Energy level of reactants has to be above a critical level for the reaction to occur
  • 67. The rate of reaction depends upon the frequency of collisions between the reactant molecules The frequency of collisions can be increased by raising the temperature
  • 68. A rise in temperature would increase: Molecular motion Frequency of collisions Rate of reaction
  • 69. The option of raising temperature is not available in living organisms In living organisms, the enzymes provide an alternate pathway for the reaction Enzymes lower the energy of activation
  • 73. Enzyme-substrate interaction The enzyme molecules are much larger than their substrates An enzyme possesses a specific binding site for its substrate(s) This site is known as the substrate site (active site) of the enzyme
  • 74. The substrate binds to the substrate site forming enzyme-substrate (ES) complex
  • 75. The binding may bring two substrates in close proximity (bond-forming distance) in the correct orientation so that a bond is formed between the two
  • 76. The binding of a substrate to the enzyme may induce a strain in the substrate As a result, a bond is broken in the substrate The substrate is split into two or more products which are released
  • 77. Substrate ‒ Enzyme ‒ Products ‒ Substrate binds to enzyme A strain occurs in the substrate; a bond is broken Substrate splits into products which are released
  • 78. On binding of two substrates to the enzyme, a chemical group may be transferred from one substrate to another
  • 79. The catalytic action of the enzyme may be exerted by: Cofactors Coenzymes Some amino acid residues in the substrate site
  • 80. In the reaction catalysed by carbonic anhydrase, the cofactor (zinc) catalyses the reaction – Zn++ H+ +HCO3 ‒ H2O H – Zn++...‒O +H+ I CO2 – Zn++ ‒ + H I +O‒C‒O +H II O – Zn++...O‒C‒O II O H H I I + H I – Zn ...O +C =O...H II O ++ ‒
  • 81. In transamination reactions, the coenzyme is involved in catalysis The coenzyme (pyridoxal phosphate) is present at the substrate site It accepts an amino group from an amino acid, and then donates it to a keto acid
  • 84. Amino acids participating in catalysis are serine, histidine, cysteine, aspartate etc In serine proteases, a serine residue at the active site catalyses proteolysis Examples of serine proteases are trypsin, chymotrypsin, thrombin etc
  • 86. The first model was proposed by Emil Fischer Also known as rigid template model A different model was later proposed by Koshland Also known as induced fit model Models of enzyme conformation
  • 87. Fischer’s model Conformation of enzymes very rigid Lock and key type of complementarity between substrate and enzyme Complementarity responsible for specificity of enzymes Lock Key
  • 89. Fischer’s model did not agree with certain experimental findings obtained later Conformation of enzyme was found to change when it combined with its substrate
  • 91. Koshland’s model conforms to known findings In the absence of substrate, complementarity between enzyme and substrate is not apparent Approach of substrate induces change in conformation of the enzyme The substrate site becomes complementary to the substrate
  • 92. The substrate binds to the enzyme, and is converted into the product Release of the product restores the enzyme to its original conformation Change in conformation of the enzyme produces ‘induced fit’
  • 94. Allosteric enzymes Some enzymes possess a site in addition to the substrate site This site is known as the allosteric site Such enzymes are known as allosteric enzymes
  • 95. Allosteric site is meant for binding of an allosteric molecule Binding of allosteric molecule changes the conformation of substrate site
  • 96. The allosteric molecule is also known as: Allosteric effector Allosteric modifier Allosteric regulator
  • 97. Some allosteric molecules: Facilitate the conformational change required for substrate binding They are known as allosteric activators (positive modifiers) They activate the enzyme
  • 98. Enzyme Substrate site Allosteric site Substrate Allosteric activator Allosteric activator binds to enzyme; substrate site changes Substrate can now bind to substrate site
  • 99. N-Acetylglutamate is an example of allosteric activator It activates carbamoyl phosphate synthetase Carbamoyl phosphate + 2 ADP + Pi CO2 +NH3 +2 ATP Carbamoyl phosphate synthetase N-Acetylglutamate ⊕
  • 100. Some allosteric regulators: Prevent the conformational change required for the binding of the substrate Such regulators are known as allosteric inhibitors (negative modifiers) An example is glucose-6-phosphate which inhibits hexokinase
  • 102. Allosteric enzymes are usually present at the start of long pathways The allosteric inhibitor is generally the product of the pathway The allosteric enzyme regulates the rate of formation of the product
  • 103. In case the product is not being utilized, it will accumulate It will inhibit the allosteric enzyme; further synthesis of the product will cease When the product is used up, the enzyme becomes free and active again
  • 104. E1 is an allosteric enzyme, and P is its allosteric inhibitor S I1 I2 I3 I4 P E1 E2 E3 E4 E5 ‒
  • 105. Factors affecting the rates of enzyme-catalysed reactions Enzyme concentration Substrate concentration Coenzyme concentration Temperature pH
  • 106. Enzyme concentration First step in an enzyme-catalysed reaction is formation of enzyme-substrate complex The enzyme-substrate complex dissociates into the enzyme and the product
  • 107. It is regenerated in its original form at the end of the reaction E +S ↔ E S ↔ E +P The enzyme may be considered to take part in the reaction
  • 108. Rate of the first reaction (formation of ES) is proportional to the product of molar concentrations of E and S Rate of formation of ES  [E] [S] Rate of the second reaction (formation of E and P) is proportional to molar concentration of ES Rate of formation of E and P  [ES]
  • 109. Therefore, the rate of the overall reaction is proportional to the enzyme concentration But this is true only if enough substrate is available to combine with the enzyme
  • 110. Rate of reaction should be proportional to substrate concentration also But this is possible only if enough enzyme is available to bind the substrate However, the availability of enzymes in the cells is limited Substrate concentration
  • 111. When the substrate concentration rises, initially the velocity of the reaction rises proportionately But later the rise in velocity becomes less until a maximum velocity (Vmax) is reached
  • 112. Vmax v [S] Vmax 2 Km Plot between substrate concentration and velocity
  • 113. At Vmax, all the enzyme molecules are saturated with the substrate The velocity cannot increase further if the substrate concentration is raised The substrate concentration at which the velocity is half of Vmax is known as Michaelis constant (Km) of the enzyme
  • 114. Vmax.[S] v = Km +[S] The relationship between velocity of reaction and the substrate concentration is given by Michaelis-Menten equation
  • 115. v = Vmax. [S] Since both Vmax and Km are constant, v  [S] When the substrate concentration is very low, the sum of Km and [S] is nearly equal to Km as [S] is negligible Hence, the equation may be rewritten as: Km Km v = Vmax x [S] or
  • 116. [S] and [S] are cancelled; the equation may be rewritten as: v =Vmax Vmax.[S] v = [S] When the substrate concentration is very high, the sum of Km and [S] is nearly equal to [S] as Km is relatively negligible Hence, the equation may be rewritten as:
  • 117. Thus, when the substrate concentration is equal to Km, the velocity is half of Vmax 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: Vmax. [S] Vmax v = = 2 [S] 2
  • 118. Determination of Km Every enzyme has got a characteristic Km Determination of Km is important in: Study of enzyme kinetics Assay of enzyme activity Evaluation of enzyme inhibitors
  • 119. Plotting v versus [S] is a lengthy process Velocity has to be measured at a number of substrate concentrations The substrate concentration has to be raised until Vmax is reached
  • 120. Lineweaver and Burk devised a simple method for determination of Km Velocity is measured at a small number (5-6) of substrate concentrations A graph is plotted between the reciprocal of v and the reciprocal of [S]
  • 121. The 1/v versus 1/[S] plot is known as: Lineweaver- Burk plot Double reciprocal plot
  • 123. 1 = Km +[S] or v [S] Vmax Vmax 1 Km 1 1 =  + Michaelis-Menten equation is inverted or v Vmax.[S] Km 1 = v Vmax.[S] [S] + Vmax.[S]
  • 124. This is the equation for a straight line y (y-axis) is 1/v a (slope of the line) is Km/Vmax x (x-axis) is 1/[S] b (y-intercept) is 1/Vmax 1 = Km  1 1 + v Vmax y = a [S] Vmax x + b
  • 125. At the x-intercept (where the line meets the x-axis), the value of y =0 Therefore, at the x-intercept: ax + b = 0 or ax = – or x = – b b a
  • 126. or On substituting the values of b and a: x = 1  Km Vmax Vmax 1 Vmax or x =  Km Vmax x = Km  1
  • 127. Thus, the value of 1/[S] at the x-intercept is 1/Km, and its reciprocal will be the Km 1 [S] 1 Km 1 v 1 Vmax
  • 128. Allosteric enzymes do not follow Michaelis-Menten equation The v versus [S] plot of allosteric enzymes is sigmoidal This shows co-operative binding of substrate to the enzyme
  • 129. [S] → [S] → ↑ v ↑ v Substrate concentration vs velocity plot Normal enzyme Allosteric enzyme
  • 130. ↑ v [S] → Positive effectors shift the plot to the left, and negative effectors shift it to the right Effect of allosteric activator and inhibitor on velocity
  • 131. Kinetics of allosteric enzymes follow the Hill equation Hill plot is plotted between log v/Vmax–v and log [S] S50 of allosteric enzymes can be determined from the Hill plot S50 is the substrate concentration at which the velocity is half of Vmax
  • 132. In coenzyme-requiring reaction, coenzyme concentration of also affects the velocity Some coenzymes form an integral part of the holoenzyme molecule Other coenzymes act as co-substrates in the reaction Coenzyme concentration
  • 133. If coenzyme is an integral part of enzyme, the effect of coenzyme concentration is same as that of enzyme concentration If coenzyme acts as a second substrate, the effect of coenzyme concentration is similar to that of substrate concentration
  • 134. To see the effect of temperature, velocity of the reaction is measured at different temperatures A curve is plotted between velocity and temperature A bell-shaped curve is obtained Temperature
  • 135. ↑ v Optimum temp │ Temp → Effect of temperature on velocity
  • 136. When the temperature rises, the velocity initially increases This is due to increased kinetic energy of the reactants
  • 137. A further rise in temperature leads to progressive denaturation of the enzyme The velocity begins to decrease as the enzyme gets denatured The reaction practically stops when the enzyme is completely denatured
  • 138. The temperature at which the velocity is the highest is known as the optimum temperature of the enzyme The optimum temperature for all human enzymes is 37°C
  • 139. The temperature coefficient (Q10) of an enzyme is the number of times the velocity rises when temperature rises by 10°C For most of the enzymes, the temperature coefficient is two This means that the velocity is doubled when the temperatures rises by 10°C
  • 140. pH A bell-shaped curve is obtained To see the effect of pH: Velocity is measured at different pH levels Velocity is plotted against pH
  • 142. A change in pH alters electrical charges on the enzyme molecules, and often on substrate molecules as well This can affect binding of the substrate to the enzyme or the catalytic activity of the enzyme or both
  • 143. At an optimum pH, the velocity of the reaction is the highest because: The electrical charges on the enzyme and the substrate are the most suitable for: Enzyme-substrate binding Catalysis
  • 144. 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
  • 145. Enzyme inhibition Catalytic activity of enzymes can be inhibited by some compounds Enzyme inhibition may be of two types: Competitive Non-competitive
  • 146. Competitive inhibition Competitive inhibition is also known as substrate-analogue inhibition The inhibitor has a close structural resemblance with the substrate Inhibitor can also bind to the substrate site of enzyme because of structural resemblance
  • 147. When inhibitor (I) binds to the enzyme, enzyme-inhibitor (EI) complex is formed However, the inhibitor cannot form the product Thus, in the presence of the inhibitor, catalytic activity of the enzyme is inhibited
  • 149. The inhibitor competes with the substrate to bind to the enzyme Hence, this type of inhibition is known as competitive inhibition
  • 151. When several molecules of substrate, inhibitor and enzyme are present together: Some enzyme molecules bind the substrate forming ES complex Some enzyme molecules bind the inhibitor forming EI complex
  • 152. Both ES and EI complexes are formed but only ES can form the product E +P E S I No P ▼ ES ◀ ► EI ▼
  • 153. Amounts of ES and EI complexes depend upon the relative concentrations of S and I If concentration of I is higher Less product will be formed More EI complex will be formed If concentration of S is higher Inhibition of enzyme will be less More ES complex will be formed
  • 154. If a Lineweaver-Burk plot is plotted in the presence of a competitive inhibitor: The y-intercept remains unchanged The x-intercept is changed
  • 155. 1/[S] → 1 1 Km K’m 1 Vmax – In the presence of inhibitor ↑ 1 v Competitive inhibition – In the absence of inhibitor
  • 156. The y-intercept is 1/Vmax which remains unchanged in the presence of competitive inhibitor The x-intercept is 1/Km which becomes less in the presence of competitive inhibitor
  • 157. Competitive inhibitors do not affect the Vmax The Vmax can be attained even in the presence of the inhibitor But more substrate is required to reach the Vmax in the presence of the inhibitor
  • 158. Efficacy of a competitive inhibitor can be assessed by measuring Km: The extent of rise in Km is a measure of efficacy of the inhibitor In the presence of the inhibitor In the absence of the inhibitor
  • 159. Competitive inhibitors of some enzymes are being used as drugs They are used to inhibit specific reactions The inhibition produces a desired pharmacological effect
  • 160. Some competitive inhibitors used as drugs are: Amethopterin and aminopterin Allopurinol Physostigmine and neostigmine Mevastatin and lovastatin
  • 161. Amethopterin and aminopterin Are structural analogues of folic acid Inhibit dihydrofolate reductase
  • 162. H2N N 1 2 N 3 4 | OH 5 6 N 7 N 8 9 10 CH2— N — | H — C — N — CH | COOH COOH | CH2 | O H CH2 || | | CH3 Folic acid Amethopterin H2N N 1 2 N 3 4 | OH 5 6 N 7 N 8 9 10 CH2—N — — C — N — CH | COOH COOH | CH 2 | O H CH2 || | | | CH3
  • 163. Dihydrofolate reductase Folate Dihydrofolate reductase Tetrahydrofolate Tetrahydrofolate is required for the synthesis of purine and thymine nucleotides NADPH +H+ NADPH+ Dihydrofolate NADPH +H+ NADPH+
  • 164. Inhibition of dihydrofolate reductase decreases the synthesis of nucleotides Decreased availability of nucleotides decreases DNA synthesis and cell division Thus, cell division is suppressed in the presence of amethopterin and aminopterin Therefore, they are used as anti-cancer drugs
  • 165. Allopurinol Is a structural analogue of hypoxanthine It inhibits xanthine oxidase
  • 167. Hypoxanthine Xanthine oxidase Xanthine Xanthine oxidase Uric acid Xanthine oxidase converts hypoxanthine into xanthine Then, it converts xanthine into uric acid
  • 168. Allopurinol is used to treat gout Gout results from over-production of uric acid Allopurinol inhibits the formation of uric acid
  • 169. Physostigmine and neostigmine Are structural analogues of acetylcholine They inhibit acetyl cholinesterase
  • 171. Physostigmine and neostigmine decrease the breakdown of acetylcholine They are used to treat myasthenia gravis, an auto-immune disorder Number of acetylcholine receptors is decreased in myasthenia gravis
  • 172. Mevastatin and Lovastatin Are structural analogues of HMG CoA Are inhibitors of HMG CoA reductase
  • 173. Mevalonate HMG CoA HMG CoA reductase Cholesterol Therefore, mevastatin and lovastatin are used as hypo-cholesterolaemic drugs Inhibition of this reaction decreases the synthesis of cholesterol HMG CoA reductase catalyses the key reaction in the synthesis of cholesterol
  • 174. Non-competitive inhibition The non-competitive inhibitors have no structural resemblance with the substrate They do not compete with the substrate for binding to the enzyme They bind to some other region of the enzyme and render it inactive
  • 175. Enzyme +Substrate + Inhibitor Enzyme +Substrate Non-competitive inhibition
  • 176. Non-competitive inhibition may be reversible or irreversible Generally, it is irreversible Examples are iodoacetamide, cyanide, p- chloromercuribenzoate, heavy metals etc
  • 177. If a Lineweaver-Burk plot is plotted in the presence of a non-competitive inhibitor: The y-intercept becomes higher The x-intercept remains unchanged
  • 178. In the presence of inhibitor In the absence of inhibitor ↑ 1 v 1/[S] → 1 Km 1 Vmax 1 V’max Non-competitive inhibition
  • 179. Non-competitive inhibitors decrease the Vmax but do not affect the Km The substrate concentration required to reach the new Vmax remains unchanged
  • 180. Chemical reactions in living organism are usually parts of some metabolic pathway A pathway consists of a series of reactions Each pathway serves some specific purpose(s) Regulation of enzymes
  • 181. Metabolic pathways need to be regulated precisely Regulation ensures adequacy of products with no wastage of raw materials Requirements of the organism keep on changing Regulatory mechanisms must be responsive to these changes
  • 182. Concentrations of enzymes Enzymes play a crucial role in the regulatory mechanisms Metabolic pathways are regulated by changing one of the following: Catalytic activity of enzymes
  • 183. Rate-limiting step in the pathway Committed step in the pathway The regulation involves one or a few “key” enzymes in a pathway The key enzyme (or regulatory enzyme) may catalyse:
  • 184. Rate-limiting step An early reaction that controls the availability of substrates for the subsequent reactions Committed step The earliest irreversible reaction unique to the pathway
  • 185. Regulation of enzyme concentration Some pathways are regulated by altering the concentrations of the key enzyme(s) If the enzyme concentration increases, the rate of reactions would increase If the enzyme concentration decreases, the rate of reactions would decrease
  • 186. Enzyme concentration can be altered by increasing or decreasing: Rate of synthesis of enzyme Regulation of enzyme synthesis is commoner Rate of breakdown of enzyme
  • 187. Regulation of enzyme synthesis Enzyme synthesis may be regulated by: Induction of enzyme synthesis Repression of enzyme synthesis Conversion of proenzyme into enzyme
  • 190. Inducer may be the substrate for the enzyme or may be a gratuitous inducer A gratuitous inducer is one which is not a substrate for the enzyme
  • 191. Inducer acts on DNA; increases expression of the gene encoding the enzyme An example is induction of key enzymes of gluconeogenesis by glucocorticoids
  • 192. Synthesis of some enzymes is regulated by repression Transcription of gene encoding the enzyme is blocked by a repressor The repressor is made up of apo- repressor and co-repressor Repression
  • 193. X X Repression of gene expression
  • 194. Apo-repressor is a protein always present in the cell When co-repressor enters or accumulates, it combines with apo- repressor to form the repressor The co-repressor is generally the product of the pathway
  • 195. An example is regulation of haem synthesis The regulatory enzyme is -aminolevulinic acid synthetase Haem is the regulator of this enzyme
  • 196. Haem acts as co-repressor; combines with aporepressor to form repressor The repressor represses the synthesis of this early enzyme in the pathway Decreased enzyme availability decreases haem synthesis
  • 197. When haem is used up, the repressor cannot be formed The repression is relieved; the enzyme synthesis re-commences This is known as derepression
  • 198. Conversion of proenzyme into enzyme Sometimes, the concentration of enzymes needs to be increased quickly For example, when food enters stomach, pepsin concentration has to be raised quickly This cannot be done by induction or derepression which are slow processes
  • 199. The enzyme is synthesized in the form of a precursor, pepsinogen Pepsinogen is an inactive proenzyme The proenzyme will not digest the mucosal proteins
  • 200. Entry of food in the stomach generates some signals These signals convert pepsinogen into pepsin The enzyme concentration is raised quickly
  • 201. Proenzyme Peptide Proteolysis Active site (masked) → Active site (exposed) → Enzyme Proteolytic activation of proenzyme Substrate
  • 202. Regulation of enzyme degradation Enzyme concentration may also be regulated by altering its breakdown Increased breakdown will decrease the concentration of the enzyme Decreased breakdown will increase the concentration of the enzyme
  • 203. Regulation of degradation is not common in higher organisms A few examples are seen in starvation in which nutrients need to be conserved Concentration of some enzymes is increased by decreasing their breakdown An example is tryptophan pyrrolase
  • 204. Regulation of catalytic activity of enzymes The key enzyme is regulated by altering its catalytic activity Enzyme concentration remains unchanged; catalytic activity is increased or decreased
  • 205. Catalytic activity of the enzyme may be altered by: Allosteric regulation of the enzyme Covalent modification of the enzyme
  • 206. Allosteric regulation This mechanism is used in some long metabolic pathways The substrate is converted into a product by a series of reactions The earliest functionally irreversible reaction is catalysed by an allosteric enzyme
  • 207. Usually, the product of the pathway is the allosteric inhibitor of the enzyme When the product accumulates, it inhibits the allosteric enzyme S I1 I2 I3 I4 P E1 E2 E3 E4 E5 ‒
  • 208. When the product is used up, the inhibition is relieved Thus, synthesis of the product is regulated according to rate of its utilization If there are a number of irreversible steps, regulation may occur at a number of steps
  • 209. Some enzymes are regulated by positive allosteric modulation (i.e. activation) An example is the first reaction of urea cycle This reaction is catalyzed by carbamoyl phosphate synthetase I (mitochondrial)
  • 210. Carbamoyl phosphate + 2 ADP + Pi N-Acetylglutamate ⊕ Carbamoyl phosphate synthetase I is an allosteric enzyme It is allosterically activated by N-acetyl- glutamate CO2 +NH3 +2 ATP Carbamoyl phosphate synthetase I
  • 211. Many enzymes are regulated by negative allosteric modulation (i.e. inhibition) An example is asparate transcarbamoylase It is an early enzyme in de novo synthesis of pyrimidine nucleotides It is inhibited by cytidine triphosphate, a product of the pathway
  • 212. A few enzymes are subject to positive as well as negative allosteric regulation Phosphofructokinase-1, a regulatory enzyme in glycolytic pathway, is subject to: Allosteric activation by AMP Allosteric inhibition by ATP
  • 213. The enzymes regulated by this mechanism can exist in two forms The two forms can be converted into each other The conversion occurs by a covalent modification of the enzyme molecule Covalent modification
  • 214. During conversion, a covalent bond is either formed or broken in the enzyme The most common covalent modification is addition or removal of phosphate
  • 215. Phosphate is usually added to or removed from a serine residue in the enzyme The phosphate group is added by a protein kinase It is removed by a protein phosphatase
  • 217. Out of the two forms of the enzyme, one is active and the other inactive The form depends upon relative activities of protein kinase and protein phosphatase These, in turn, are controlled by hormones acting through second messengers
  • 218. An example is glycogen synthetase, the regulatory enzyme of glycogenesis Its dephosphorylated form is active and the phosphorylated form is inactive
  • 220. Another example is glycogen phospho- rylase, the key enzyme of glycogenolysis Its phosphorylated form is active and the dephosphorylated form is inactive
  • 222. Some enzymes are regulated by multiple mechanisms For example, acetyl CoA carboxylase is subject to: Induction Repression Allosteric regulation Covalent modification
  • 223. A large number of enzymes are synthesized in various cells They are continuously released into circulation due to natural cell death They are continually removed from circulation by degradation or excretion Enzymes of diagnostic importance
  • 224. The circulating enzymes may be divided into two types: Functional plasma enzymes or plasma-specific enzymes Non-functional plasma enzymes or non-plasma-specific enzymes The enzymes are normally present in circulation in very low concentrations
  • 225. Functional plasma enzymes These enzymes are purposely secreted into circulation They perform specific catalytic functions in plasma Examples are lipoprotein lipase, blood clotting factors, complement proteins etc
  • 226. Non-functional plasma enzymes These enzymes do not perform any function.in plasma These are intracellular enzymes which enter the circulation when the cells in which they are synthesized die
  • 227. Non-functional plasma enzymes or non- plasma-specific enzymes These enzymes do not perform any function.in plasma These are intracellular enzymes which enter the circulation when the cells in which they are synthesized die
  • 228. When cell death is occurring at normal rate, non-functional enzymes are released in very small amounts Their concentrations in plasma remain very low
  • 229. If the rate of cell death increases, these enzymes are released in large amounts Their concentrations in plasma can rise many times above normal
  • 230. A non-functional plasma enzyme can pin- point the site of the disease IF It has a selective tissue distribution OR If its concentration is far higher in some tissues than elsewhere in the body
  • 231. Thus, the enzymes of diagnostic importance are: The non-functional plasma enzymes ‒ Having a selective tissue distribution
  • 232. Plasma enzymes that are established diagnostic tools: • Lactate dehydrogenase (LDH) • Transaminases (GOT and GPT) • Creatine kinase (CK) • Gamma-glutamyl transpeptidase (GGT) • Alkaline phosphatase (ALP) • Acid phosphatase (ACP) • Amylase • Lipase • Ceruloplasmin
  • 233. Lactate dehydrogenase (LDH) Catalyses interconversion of pyruvate and lactate Tissue distribution very wide Concentration very high in myocardium, muscles and liver
  • 234. Plasma LDH rises in: Myocardial infarction Viral hepatitis Muscle injuries
  • 235. In myocardial infarction: Rise begins 24 hours after infarction Peak value is reached in about three days Level returns to normal in about a week
  • 236. Transaminases The two most important are GOT and GPT GOT is glutamate oxaloacetate transaminase GPT is glutamate pyruvate transaminase
  • 237. GOT is also known as aspartate aminotransferase (AST) GPT is also known as alanine aminotransferase (ALT)
  • 238. GOT and GPT are present in high concentrations in: Liver Muscles Myocardium
  • 239. Serum GOT and GPT are raised in: Myocardial infarction Viral hepatitis Muscle injuries
  • 240. Concentration of GOT is higher than that of GPT in myocardium while the situation is reverse in liver Therefore Rise in plasma GOT is more in myocardial infarction and that in GPT is more in viral hepatitis
  • 241. Creatine kinase (CK) Also known as creatine phosphokinase (CPK) Catalyses interconversion of creatine and creatine phosphate Creatine +ATP ↔ Creatine~ ℗+ADP
  • 242. CK is present in: Brain Muscles Myocardium
  • 243. Serum CK is raised in: Myocardial infarction Myopathies Muscle injuries
  • 244. Rise begins within 3-6 hours after MI Peak is reached in 24 hours Returns to normal in three days Specific and early indicator of MI Serum CK in myocardial infarction (MI)
  • 245. Enzyme level Upper limit of normal 0 1 2 3 Days 4 5 6 7 CK GOT LDH
  • 246. Begins to rise in Reaches peak in Returns to normal in Specificity Myoglobin 1-3 hrs 4-6 hrs 18-24 hrs Low Cardiac troponin T 4-6 hrs 18-36 hrs 5-15 days High Cardiac troponin I 4-6 hrs 12-24 hrs 5-10 days High Non-enzyme markers of myocardial infarction
  • 247. Gamma-glutamyl transpeptidase (GGT) Transfers the -glutamyl residue of glutathione to other substrates Serum level increases in most of the liver diseases Is an early indicator of alcoholic hepatitis
  • 248. Alkaline phosphatase (ALP) ALP is a group of enzymes The group hydrolyses organic phosphate esters Its optimum pH is in alkaline range
  • 249. ALP is released in circulation mainly from bones and liver Smaller amounts are released from intestines and placenta Liver excretes ALP in bile
  • 250. A marked rise in plasma ALP occurs in obstructive jaundice Smaller elevations occur in: Viral hepatitis Rickets Hyperparathyroidism Osteosarcoma Bony metastases
  • 251. Acid phosphatase (ACP) ACP is a group of enzymes The group hydrolyses organic phosphate esters Its optimum pH is in acidic range
  • 252. The main source of circulating ACP is the prostate gland Serum ACP is elevated in metastatic carcinoma of prostate
  • 253. Amylase A digestive enzyme synthesized in the pancreas and the parotid gland Sharp elevation of serum amylase occurs in acute pancreatitis A smaller elevation occurs in acute parotitis
  • 254. Lipase A lipolytic enzyme released into circulation from the pancreas Serum lipase rises in acute pancreatitis
  • 255. Ceruloplasmin A copper-containing protein having ferroxidase activity Absent or very low in serum in Wilson’s disease (hepatolenticular degeneration)
  • 256. Isoenzymes Multiple molecular forms of the same enzyme All catalyse the same reaction They differ slightly in physical, chemical and immunological properties
  • 257. Isoenzymes possess quaternary structure They are made up of two or more sub- units that are different from each other The subunits have slightly different primary structures
  • 258. Isoenzymes usually differ in Km and Vmax Their regulation may be different This helps in fine-tuning of metabolism
  • 259. Isoenzymes can be separated by: Electrophoresis Chromatography Immunochemical methods
  • 260. The tissue distribution of isoenzymes is highly specific Measurement of isoenzymes can be of great diagnostic importance
  • 261. Isoenzymes of diagnostic importance include: Lactate dehydrogenase Creatine kinase Alkaline phosphatase
  • 262. Lactate dehydrogenase H subunit M subunit First enzyme shown to exist in the form of five isoenzymes by Markert (1957) The enzyme is a tetramer made up of two types of subunits – H and M
  • 263. • HHHH • HHHM • HHMM • HMMM • MMMM The subunits can form five different tetramers (isoenzymes): or LD1 or LD2 or LD3 or LD4 or LD5 or LDH1 or LDH2 or LDH3 or LDH4 or LDH5
  • 264. The LD isoenzymes in plasma can be separated by electrophoresis The normal pattern of LD isoenzymes in serum is LD2 >LD1 >LD3 >LD4 >LD5
  • 265. The predominant isoenzymes in myocardium are LD1 and LD2 Both are raised in myocardial infarction The rise in LD1 is greater than that in LD2 Hence, the plasma LD isoenzyme pattern becomes LD1 >LD2 >LD3 >LD4 >LD5
  • 266. LD5 is the predominant isoenzyme in liver Therefore, LD5 is raised in viral hepatitis
  • 267. Creatine kinase B subunit M subunit A dimer made up of two types of subunits The subunits are – B and M
  • 268. Three different dimers (isoenzymes) can be formed from these two subunits: • BB or CK1 or CK-BB • MB or CK2 or CK-MB • MM or CK3 or CK-MM
  • 269. CK-MB is commonly measured by immuno- inhibition Serum is treated with anti-M subunit antibody CK-MM is inhibited The residual enzyme is taken to be CK-MB as CK-BB is negligible
  • 270. Normal CK isoenzyme pattern in plasma CK-MB CK-BB CK-MM
  • 271. The major isoenzyme in myocardium is CK-MB In plasma, CK-MB is less than 3% of total CK CK-MB is raised in myocardial infarction
  • 272. CK-BB, CK-MB and CK-MM are present in cytosol A different CK is present in mitochondria – mitochondrial CK (CK-MT or CK-Mi)
  • 273. CK-MT has two isoforms: CK-MT1 and CK-MT2 CK-MT1 is ubiquitous CK-MT2 is present in skeletal and heart muscle
  • 274. CK-MT can exist as a dimer or an octamer The dimeric and octameric forms are inter-changeable
  • 275. CK-MT1 and CK-MT2 are encoded by different genes Thus, there are four genes for CK subunits These are CK-M, CK-B, CK-MT1 and CK-MT2 genes
  • 276. CK-M and CK-B genes encode the cytosolic enzyme CK-MT1 and CK-MT2 genes encode the mitochondrial enzyme CK-MT1 and CK-MT2 isoenzymes have no diagnostic importance
  • 277. Bone, liver , intestine and placenta form different isoenzymes ALP isoenzymes are commonly separated by electrophoresis Liver isoenzyme moves the fastest and occupies the same position as 2-globulin Alkaline phosphatase
  • 278. The bone ALP closely follows the liver ALP The placental isoenzyme follows the bone isoenzyme The intestinal isoenzyme is the slowest moving
  • 279. The liver ALP is raised in liver cancer and biliary obstruction The bone ALP is raised in bone cancers and Paget’s disease The placental and intestinal isoenzymes have no diagnostic importance
  • 280. Two atypical ALP isoenzymes are seen in some cancers These are Regan isoenzyme and Nagao isoenzyme Regan and Nagao isoenzymes resemble the placental isoenzyme
  • 281. Regan isoenzyme is raised in cancer of breast, lungs, colon, uterus and ovaries Nagao isoenzyme is raised in germ cell cancer of the testes
  • 282. Assay of enzymes Several enzymes present in circulation help in diagnosis of diseases For this, we need to measure serum levels of these enzymes Sometimes, such measurement is required for academic purpose
  • 283. Enzyme concentrations in serum are very minute Isolation and purification of enzymes is difficult and time-consuming Therefore, direct measurement of enzyme concentrations is very difficult
  • 284. Enzyme concentrations are measured indirectly Velocity of the enzyme-catalyzed reaction is measured Conditions are such that rate of reaction is proportional to the enzyme concentration
  • 285. The reaction is carried out in a fixed- temperature water-bath or an incubator Optimum pH is maintained by using a buffer Substrate concentration is kept constant and high Rate of reaction in such conditions is proportional to the enzyme concentration
  • 286. The rate of the reaction can be determined by measuring: The rate of disappearance of the substrate Rate of appearance of the product
  • 287. In endpoint methods: The reaction is carried out for a fixed period Initial and final concentrations of the substrate or the product are measured
  • 288. In kinetic methods, the concentration of the substrate or the product is measured at regular intervals for a brief period The result in both the methods is expressed in arbitrary units of enzyme activity rather than enzyme concentration
  • 289. Many enzymes are used as tools in diagnostic and research laboratories Glucose oxidase and peroxidase are used for measuring glucose concentration Hexokinase and glucose-6-phosphate dehydrogenase are also used for measuring glucose concentration Enzymes as laboratory tools
  • 290. 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
  • 291. Urease is used for measurement of urea concentration Uricase is used for measuring uric acid concentration Peroxidase and alkaline phosphatase are used to label antibodies in ELISA
  • 292. A number of enzymes are used in recombinant DNA technology e.g. DNA ligase Terminal transferase S1 nuclease Reverse transcriptase Taq polymerase Restriction endonucleases
  • 293. Some human, animal, plant and microbial enzymes are used as drugs also Diastase, papain, pepsin, chymotrypsin etc are used to aid digestion Amylase, lipase and proteases are used in the treatment of pancreatic insufficiency Enzymes as drugs
  • 294. Serratiopeptidase is a bacterial proteolytic enzyme It is used to remove dead tissue from the site of inflammation to accelerate healing It is also used to reduce inflammation, oedema and pain
  • 295. catalyses hydrolysis of Hyaluronidase hyaluronic acid Hyaluronidase injections are used to facilitate delivery of other injectable drugs
  • 296. Asparaginase is used in the chemotherapy of leukaemia Leukaemic cells are deficient in asparagine synthetase For their asparagine requirement, they are dependent on pre-formed asparagine
  • 297. converts asparagine into Asparaginase aspartate This deprives the leukaemic cells of an essential nutrient
  • 298. Thrombolytic drugs used to clear blockage of blood vessels are: Urokinase Streptokinase Tissue plasminogen activator