Assignment title:

Enzyme kinetics and Enzyme Inhibition

Submitted to:

Dr. Nazir Ahmad

Submitted by:

Nehal Bilal
Roll no.
0152-BH-CHEM-16
Course Code:
Chem-4234

Contents
Introduction:...............................................................................................................................................3

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General Principle:.......................................................................................................................................3
Explanation:................................................................................................................................................3
Transient state:...........................................................................................................................................4
The Michaelis Menten Kinetics:.................................................................................................................6
The turnover number:................................................................................................................................7
KM:...............................................................................................................................................................7
Kcat:..............................................................................................................................................................7
Vmax:.............................................................................................................................................................7
Conclusions:................................................................................................................................................7
Inhibitors:....................................................................................................................................................8
Reversible inhibitors:..................................................................................................................................8
Competitive inhibitors:...........................................................................................................................8
Uncompetitive inhibitor:......................................................................................................................11
Non-competitive inhibitor:...................................................................................................................13
Allosteric Reactions:.................................................................................................................................14
Irreversible inhibitors:..............................................................................................................................15
References:...............................................................................................................................................16

Enzyme Kinetics:
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Introduction:
Study of chemical reaction catalyzed by enzymes are called enzymes kinetic and by using kinetic
we can find the effect of enzymes catalysis kinetics of enzymes show its activity times rates of change
with substrate. Enzymes are usually protein and are called biocatalyst because one body work only on
enzymes activity and reaction substrate bind to the active rate of enzymes and transformed into
product.

Mechanism of enzyme bindings are off two types.

 Single substrate binding mechanism.

 Double substrate binding mechanism.

Example of signal substrate is triphosphate isomerase and when enzyme bind with multiple substrate
then the sequence of binding and product formation and can be done by enzyme kinetic.

E + S ↔ ES → E + P

General Principle:
Before uncatalysed reaction and after catalyzed reaction rate remain same. Enzymes do not
change the equilibrium. The rate of reaction increase with the substrate concentrations because more
the substrate cover the active site of enzymes more will be the product but if substrate concentration
increases beyond the certain limit then saturation point will come where reaction rate stops increasing
and become constant. KM is the substrate constant in half way between two states (Normal and
Saturated).

Explanation:
The reaction of enzyme catalysis is

K1 K2

E + S ↔ ES → E + P
K-1
This equation is according to the transition state theory.

The rate of formation of product is

V = d[P]/ dP = K2 [ES]

Rate of formation of enzyme substrate complex is

d[ES]/ dt = K1 [E][S] – K-1[ES]- K2[ES]

Here, the use of minus sign for both values from right side means formation rate is always positive and
destruction rate is always negative. So, we can say rate of formation is equal to rate of production.
The rate determining step here is

K1

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 E + S ↔ ES
K-1
At equilibrium:

k1 [ES] = k-1 [ES]

Where

KD = k1/k-1 = [E][S]/[ES]

Here, KD is the dissociated rate constant for [ES] complex.

Transient state:
Transient state is that state in which we study elementary steps of enzyme catalyzed reaction,
where the concentration of [ES] complex does not change.

d[ES]/dt = 0

This is the graph between concentration and time which shows transient phase where concentration
remain constant after certain limit substrate. We have applied steady state approximation here and
then

[E] = [E]T + [ES]

Combine equation 1, 2, and 3. We get

K1([ET – Es])[S] = (K-1 + K2)[ES]

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The Michaelis Menten Kinetics:
Enzyme catalysis show linear state after Vmax because at large concentration
enzyme catalysis become saturable. The rate of reaction become dependent on ES complex and show
zero order in case of Unimolecular reaction. K M is the substrate concentration when Vmax will half of
maximum.

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Here, KM denotes the substrate concentration where V 󠅮̥is half of the maximum.

When KD decrease then substrate concentration increases and k M can be measured by affinity when K2 is
less less than K1 and these parameters K1 and Vmax are used to compare enzyme activity.

Linear form of Michaelis Menten Equation:

Most of the scientists tried to make linear form of Michaelis Menten graph because it
was difficult to estimate KM and Vmax by non-linear graph. So, Lineweaver-Burk plot, Eadle Hotstee
diagram, and the Hans Hoff Diagram is used to see the linearity of Michaelis Menten graph.this is done
by taking reciprocal of Michaelis Menten equation from both sides, this is also known as double
reciprocal. The linearity shows that this graph is according to straight line graph. Y= mx+c andwhere
equals to 1/Vmax an x is the intercept of graph representing slope.

The small errors on low [S] leads to large errors in 1/V̥.

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The turnover number:
The turnover number is that one which shows how many molecules of catalyst
convert substrate into problem. Kcat is the turnover rate constant which can be defined as reaction of
enzyme on catalysts per second.

V̥ = Vmax [S]/KM + [S] Kcat = Vmax/ [E]T = K2

For Michaelis Menten Equation K2 = Kcat

Where [S] is less than KM very little ES is formed and [E] = [E]T.

V̥ is approximately equal to K2/KM [E][S] na this is approximately equals to K cat/KM [E][S].

Kcat/KM is the measures of catalytic efficiency.

Now, we talked about KM, Kcat and Vmax.

KM:
This shows that how strongly the enzyme binds to the substrate, higher value of K M shows the strength
of enzyme is low.

Kcat:
This show how fast the enzyme is catalyzed, high value of K cat shows large enzyme activity.

Vmax:
Vmax is related to Kcat as Vmax = Kcat [ET].

Conclusions:
 Kcat/ KM ratio shows the enzyme efficiency.
 The turnover rate of enzyme Kcat = Vmax/[ET].
 KM is the useful indicator to check the affinity of enzyme with the substrate.
 KM must be low to a good enzymatic reaction.
 Kcat must be at high rate it means that the reaction catalysis rate is high.

Enzyme Inhibition:
Inhibitors:
Inhibitors are those compounds which reduce or retard the rate of enzyme catalysis. These compound
decrease the efficiency of enzymes and destroy the active site or other side opposite to the active site of
enzyme.

There are two types of inhibitors.

 Reversible inhibitors

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  Irreversible inhibitors

Reversible inhibitors:
These are those compounds which can separate from the enzyme and are flexible in nature. These
includes

 Competitive inhibitors
 Non-competitive inhibitors
 Uncompetitive inhibitors

Competitive inhibitors:
These inhibitors are same as that of substrate and bind to the active site of the
substrate and block the site for further reaction.

E+ S → ES→ E+ P
This equation is substrate enzyme equation while now enzyme inhibitor equation is

E+ I → EP → NO PRODUCT
This show that inhibitor block the active site and no further reaction occur.

Now, when competitive inhibitor binds it block the active site of the enzyme but when we plot the data
it shows that the straight line of graph meet at the end on x-axis having same intercept.

1 1
The values are = α k m ❑ V max + +1/V max
( )
V́ ❑ S

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Example of the competitive inhibitor is shown in the diagram given below.

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Uncompetitive inhibitor:
Uncompetitive inhibitor are those inhibitors which can bind to the ES complex of enzyme and block the
formation of product.

E+ S → ES→ E+ P
And inhibitor when binds gives the reaction

E+ S → ES+ I → ESI → NO PRODUCT

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And when we plot the graph of non-competitive inhibitor then shows change in intercept without the
change of slope.

Example:

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Non-competitive inhibitor:
These are also called mixed inhibitors which resemble both the competitive and uncompetitive
inhibitors. There are two possibilities of this reaction, one is when the ES again form and the other is
when EI complex form and stop the product formation. And the graph of non-competitive inhibitor
shows that its slope value as well as its intercept changes after reaction.

Example:

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Allosteric Reactions:
Allosteric reactions are those reactions in which inhibitors bind with the enzyme other
than the active site of substrate and change the conformational shape of the enzyme so enzyme cannot
further do any reaction.

Examples are:

 Hemoglobin (Not Myoglobin)

 Gene regulating protein

 Conformational shape changes due to allosteric site.

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  Active site changes because of the allosteric reaction.
 Substrate cannot bind to the enzyme.

Comparison between competitive. Non-competitive and uncompetitive inhibitors.

Irreversible inhibitors:
Irreversible inhibitors are those which cannot detach from the enzyme. And are used as the suicide
inactivators.

 These are sometime called mechanism based inhibitors.

 They do the first reaction normal then block the sites permanently and stop second reactions.
 They are used to produce drugs.
 They usually formed covalent linkage.

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References:
 Wrigton MS, Ebbing DD (1993) “General Chemistry”.
 .J. Harglove M.S. (2013) “Enzyme kinetics” in essentials of biochemistry.
 Single molecule enzymology, “The General of biological” pp 274.
 Tech Me Physiology, “Enzyme Kinetics” https://teachmephysiology.com/basics/enzyme-
activity/enzyme-kinetics/
 Enzyme Kinetics, Elseiver’s integrated review pharmacology (second edition), 2012.

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