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
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
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
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
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
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
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
70.
71.
72.
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
85.
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
97. Some allosteric molecules:
Facilitate the conformational
change required for substrate binding
They are known as allosteric
activators (positive modifiers)
They activate the enzyme
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
101.
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
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]
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
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
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
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
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
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
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
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
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
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
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
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)
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
256. Isoenzymes
Multiple molecular forms of the same
enzyme
All catalyse the same reaction
They differ slightly in physical, chemical
and immunological properties
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
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
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