Enzymes

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Enzymes
R.C. Gupta Professor and Head Dept. of
Biochemistry National Institute of Medical
Sciences Jaipur, India
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E M B - R C G
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E M B - R C G
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E M B - R C G
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E M B - R C G
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Definition
E M B - R C G
But this definition is not entirely correct
Some RNA molecules (ribozymes) have been found to
catalyze some reactions
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E M B - R C G
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Enzyme specificity
E M B - R C G
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E M B - R C G
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E M B - R C G
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E M B - R C G
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E M B - R C G
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E M B - R C G
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E M B - R C G
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EMB-RCG
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E M B - R C G
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E M B - R C G
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E M B - R C G
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Coenzymes and cofactors
Some enzymes require a non-protein substance for
their catalytic activity
E M B - R C G
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
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E M B - R C G
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E M B - R C G
Apoenzyme Coenzyme ? Holoenzyme
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COENZYME
APOENZYME
HOLOENZYME
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E M B - R C G
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E M B - R C G
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E M B - R C G
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E M B - R C G
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E M B - R C G
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In the first reaction, the coenzyme ATP acts
as a second substrate and donates a phosphate
group
In the second reaction, the coenzyme NAD acts a
second substrate and accepts the hydrogen atoms
EMB-RCG
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E M B - R C G
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E M B - R C G
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E M B - R C G
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E M B - R C G
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E M B - R C G
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Enzyme nomenclature and classification
E M B - R C G
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E M B - R C G
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Nomenclature was modified further, to include the
name of the substrate followed by the type
of reaction ending with -ase
E M B - R C G
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
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E M B - R C G
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E M B - R C G
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The IUB name shows that
This enzyme acts on L-glutamate
E M B - R C G
NAD or NADP is required as a co-substrate
Type of reaction is oxidoreduction i.e.
L-glutamate is oxidised and the co-substrate is
reduced
The amino group of L-glutamate is released as
ammonia
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E M B - R C G
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E M B - R C G
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E M B - R C G
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E M B - R C G
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E M B - R C G
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E M B - R C G
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E M B - R C G
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E M B - R C G
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E M B - R C G
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E M B - R C G
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E M B - R C G
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E M B - R C G
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E M B - R C G
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E M B - R C G
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E M B - R C G
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Mechanism of action of enzymes
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The greater the frequency of collisions
between the reactant molecules, the greater
will be the rate of reaction
E M B - R C G
The frequency of collisions can be increased
by raising the temperature
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E M B - R C G
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E M B - R C G
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E M B - R C G
In living organisms, the enzymes provide an
alternate pathway for the reaction
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Enzyme-substrate interaction
E M B - R C G
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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
E M B - R C G
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E M B - R C G
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On binding of two substrates to the enzyme, a
chemical group may be transferred from one
substrate to another
E M B - R C G
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E M B - R C G
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In the reaction catalysed by carbonic anhydrase,
the cofactor (zinc) catalyses the reaction
H2O
? Zn
H HCO3?
CO2
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The coenzyme (pyridoxal phosphate) is present at
the substrate site
E M B - R C G
It accepts an amino group from an amino acid, and
then donates it to a keto acid
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Coenzyme
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In serine proteases, a serine residue at the
active site catalyses proteolysis
E M B - R C G
Examples of serine proteases are trypsin,
chymotrypsin, thrombin etc
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Models of enzyme conformation
E M B - R C G
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EMB-RCG
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Fischers model did not agree with certain
experimental findings obtained later
E M B - R C G
Conformation of enzyme was found to change when
it combined with its substrate
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Koshlands 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
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Change in conformation of the enzyme produces
induced fit
The substrate binds to the enzyme, and is
converted into the product
Release of the product restores the enzyme to its
original conformation
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E M B - R C G
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E M B - R C G
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The allosteric molecule is also known as
Allosteric effector
E M B - R C G
Allosteric modifier
Allosteric regulator
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E M B - R C G
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E M B - R C G
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E M B - R C G
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Allosteric enzymes are usually present at the
start of long pathways
The allosteric inhibitor is generally the product
of the pathway
E M B - R C G
The allosteric enzyme regulates the rate of
formation of the product
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If the product is not being utilized, it
will accumulate
E M B - R C G
It inhibits the allosteric enzyme, and further
synthesis of the product ceases
When the product is used up, the enzyme becomes
free and active again
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E1 is an allosteric enzyme, and P is its
allosteric inhibitor
EMB-RCG
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E M B - R C G
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Enzyme concentration
E M B - R C G
EMB-RCG
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E S ? E S ? E P
EMB-RCG
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• Rate of the first reaction leading to
  formation of ES is proportional to the
  product of molar concentrations of E and S
  
  
  
  
  

E M B - R C G
Rate of formation of ES µ E S
Rate of the second reaction leading to
formation of E and P is proportional to molar
concentration of ES
Rate of formation of E and P µ ES
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Therefore, the rate of the overall reaction
is proportional to the enzyme concentration
E M B - R C G
But this is true only if enough substrate
is available to combine with the enzyme
EMB-RCG
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Substrate concentration
E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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Plot between substrate concentration and velocity
EMB-RCG
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At Vmax, all the enzyme molecules are saturated
with substrate, and velocity cannot increase
further if the substrate concentration is raised
E M B - R C G
The substrate concentration at which the velocity
is half of Vmax is known as the Michaelis
constant (Km) of the enzyme
EMB-RCG
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The relationship between the velocity of the
reaction and the substrate concentration can be
expressed by Michaelis-Menten equation
E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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Determination of Km
E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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Michaelis-Menten equation
EMB-RCG
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EMB-RCG
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y a x b
EMB-RCG
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E M B - R C G
EMB-RCG
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or
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EMB-RCG
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E M B - R C G
EMB-RCG
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Effect of allosteric activator and inhibitor on
velocity
EMB-RCG
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E M B - R C G
EMB-RCG
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Coenzyme concentration
E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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Temperature
E M B - R C G
EMB-RCG
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Optimum temp
? v
Temp ?
Effect of temperature on velocity
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E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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pH
E M B - R C G
EMB-RCG
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Optimum pH
? v
pH ?
Effect of pH on velocity
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E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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Enzyme inhibition
E M B - R C G
EMB-RCG
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Competitive inhibition
E M B - R C G
EMB-RCG
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Substrate
Products ??
Inhibitor
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E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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The relative amounts of ES and EI complexes
depend upon the relative concentrations of the
substrate and the inhibitor
E M B - R C G
If the inhibitor concentration is higher, more EI
complex will be formed resulting in decreased
formation of the product
If the substrate concentration is higher, more ES
complex will be formed, and the inhibition will
be less
EMB-RCG
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If a Lineweaver-Burk plot is plotted in the
presence of competitive inhibitor, the
y-intercept (1/Vmax) remains unchanged
E M B - R C G
However, the apparent Michaelis constant (Km) is
higher (1/Km is lower) in the presence of
competitive inhibitor
EMB-RCG
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Competitive inhibitors do not affect the Vmax
The Vmax can be attained even in the presence of
the inhibitor
E M B - R C G
But more substrate is required to reach the Vmax
in the presence of the inhibitor
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E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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Folic acid
COOH
H N
N
N
2

1
8
2
CH
7
2

N
3
6
CH
O
H
2
5
4



9
10
N
CH N
C N CH

2


OH
CH3
COOH
CH3
Amethopterin
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• Inhibition of dihydrofolate reductase
  decreases the availability of nucleotides
• If nucleotides are not available, DNA synthesis
  and cell division are inhibited
• Therefore, amethopterin and aminopterin are
  used in cancer to suppress cell division

E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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EMB-RCG
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EMB-RCG
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E M B - R C G
EMB-RCG
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Non-competitive inhibition
E M B - R C G
EMB-RCG
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Enzyme Substrate
Enzyme Substrate
Inhibitor
Non-competitive inhibition
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E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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Enzymes of diagnostic importance
E M B - R C G
EMB-RCG
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Functional plasma enzymes or plasma-specific
enzymes
E M B - R C G
EMB-RCG
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Non-functional plasma enzymes or
non-plasma-specific enzymes
E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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Thus, it is the non-functional plasma
enzymes having a selective tissue
distribution which can give information of
diagnostic importance
E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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Concentration of GOT is higher than that
of GPT in myocardium while the situation
is reverse in liver
E M B - R C G
Therefore
Rise in plasma GOT is more in myocardial
infarction and that in GPT is more in
viral hepatitis
EMB-RCG
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Creatine ATP ? Creatine ? ADP
EMB-RCG
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E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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Serum CK in myocardial infarction
E M B - R C G
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Non-enzyme markers of myocardial infarction
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E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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Isoenzymes
E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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The tissue distribution of isoenzymes is
highly specific
E M B - R C G
Measurement of isoenzymes can be of great
diagnostic importance
EMB-RCG
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E M B - R C G
EMB-RCG
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Lactate dehydrogenase
E M B - R C G
H subunit
M subunit

EMB-RCG
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E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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Creatine kinase
A dimer made up of two types of subunits
The subunits are B and M
EMB-RCG
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• BB or CK1 or CK-BB
• MB or CK2 or CK-MB
• MM or CK3 or CK-MM

EMB-RCG
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E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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Alkaline phosphatase
E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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Regulation of enzymes
E M B - R C G
EMB-RCG
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E M B - R C G
The rate-limiting step in the pathway
The committed step in the pathway
EMB-RCG
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E M B - R C G
EMB-RCG
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Regulation of enzyme concentration
E M B - R C G
EMB-RCG
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Regulation of enzyme synthesis
E M B - R C G
EMB-RCG
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Induction
E M B - R C G
EMB-RCG
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Constitutive enzymes Inducible enzymes
Continuously synthesized Synthesized only when required
Always present in the cell Synthesized when inducer enters the cell
E M B - R C G
EMB-RCG
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Inducer may be the substrate for the enzyme or
may be a gratuitous inducer
E M B - R C G
EMB-RCG
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The inducer acts on DNA, and increases the
expression of the gene that encodes the enzyme
E M B - R C G
An example is induction of key enzymes of
gluconeogenesis by glucocorticoid hormones
EMB-RCG
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Repression
Synthesis of some enzymes is regulated by
repression
E M B - R C G
Transcription of gene encoding the enzyme is
blocked by a repressor
The repressor is made up of apo-repressor and
co-repressor
EMB-RCG
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Apo-repressor is a protein always present in
the cell
E M B - R C G
When co-repressor enters or accumulates in the
cell, it combines with apo-repressor to form the
repressor
The co-repressor is generally the product of
the pathway
EMB-RCG
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An example is regulation of haem synthesis
by d-aminolevulinic acid synthetase
E M B - R C G
Haem acts as co-repressor, and represses the
synthesis of this early enzyme in the pathway
When the product is used up, the repression is
relieved (derepression)
EMB-RCG
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Conversion of proenzyme into enzyme
E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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Regulation of enzyme degradation
E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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Regulation of catalytic activity of enzymes
Enzyme concentration remains unchanged but its
catalytic activity is altered
E M B - R C G
The catalytic activity may be altered by
Allosteric regulation of the enzyme
Covalent modification of the enzyme
EMB-RCG
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Allosteric regulation
E M B - R C G
EMB-RCG
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T
E1 E2 E3 E4 E5
S I1 I2
I3
I4
P
EMB-RCG
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E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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A few enzymes are subject to positive as well as
negative allosteric regulation
E M B - R C G
Phosphofructokinase-1, a regulatory enzyme in
the glycolytic pathway, is subject to
Allosteric activation by AMP
Allosteric inhibition by ATP
EMB-RCG
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Covalent modification
E M B - R C G
EMB-RCG
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Phosphate is usually added to or removed from a
serine residue in the enzyme
A protein kinase adds phosphate, and a protein
phosphatase removes phosphate
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One form, either phosphorylated or
dephospho-rylated, is active and the other
is inactive
E M B - R C G
Whether the enzyme is active or inactive depends
upon the relative activities of protein kinase
and protein phosphatase
These, in turn, are controlled by hormones
acting through second messengers
EMB-RCG
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An example is glycogen synthetase ? active in the
dephosphorylated form and inactive in the
phosphorylated form
E M B - R C G
Another example is glycogen phosphorylase ?
inactive in the dephosphorylated form and active
in the phosphorylated form
EMB-RCG
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Assay of enzymes
E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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Enzymes as laboratory tools
E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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Enzymes as drugs
E M B - R C G
EMB-RCG
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E M B - R C G
EMB-RCG
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Thank you