ENZYME KINETICS

Dr. Javed Anver Qureshi

Professor of Biochemistry,
Institute of Molecular Biology and Biochemistry (IMBB)
The University of Lahore
 Enzymes
Enzymes are both proteins and biological catalysts (biocatalysts)

Often called organic thermo-labile catalysts that accelerate the rate of

reaction biological reaction without being consumed in the reaction.

Alters the rate of reaction in biological process.

Enzymes not alter the equilibrium

They are high molecular weight compounds , made up mainly of

chains of amino acids linked together by peptide bonds.

Nature of Enzymes
All enzymes are protein in nature except ribozymes (RNA in nature).
 Enzyme Kinetics
The word kinetics is derived from the Greek word kinetos,
which means “moving”.
Enzyme kinetics is the study of the chemical reactions that
are catalysed by enzymes.
In enzyme kinetics, the reaction rate is measured and the
effects of varying the conditions of the reaction is
investigated.
Studying an enzymes kinetics in this way can reveal the
catalytic mechanism of this enzyme
Enzyme kinetics is concerned with the influence of
enzymes on biochemical reaction rates.
 Michaelis-Menten derivation for simple steady-state kinetics

The Michaelis-Menten equation is a mathematical

model that is used to analyze simple kinetic data. The
model has certain assumptions, and as long as these
assumptions are correct, it will accurately model your
experimental data. The derivation of the model will highlight
these assumptions.
Michaelis-Menten derivation for simple steady-state kinetics
In an enzyme catalyzed reaction the substrate initially forms a
reversible complex with the enzyme (i.e. the enzyme and substrate
have to interact for the enzyme to be able to perform its catalytic
function). The standard expression to show this is the following:

K1: The binding of the enzyme to the substrate forming the

enzyme substrate complex (E S)
K-1: The dissociation of the enzyme-substrate complex
(ES)to free enzyme and substrate ( E + S)
K2: Catalytic rate; the catalysis reaction producing the
final reaction product (P) and regenerating the free
enzyme (E ) . This is the rate limiting step.
K-2 Formation of ES from E + P
ASSUMPTION #1:
There is no product present at the start of the kinetic analysis
 Therefore, as long as initial reaction rates are monitored ,the
reverse reaction of E+P going to ES can be ignored

ASSUMPTION #2:
 During the reaction an equilibrium condition is established for
the binding and dissociation of the Enzyme and Substrate
(Briggs-Haldane assumption)
 Thus, the rate of formation of the ES complex is equal to
the rate of dissociation plus breakdown
ASSUMPTION #3:
 [E] << [S]
 The enzyme is a catalyst, it is not destroyed and can be recycled,
thus, only small amounts are required
 The amount of S bound to E at any given moment
is small compared to the amount of free S
 It follows that [ES] << [S] and therefore [S] is constant during the
course of the analysis (NOTE: this assumption requires that the
reaction is monitored for a short period, so that not much S is
consumed and [S] does not effectively change - see next
assumption)

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ASSUMPTION #4:
 Only the initial velocity of the reaction is measured
 [P] = 0 (reverse E + P reaction can be ignored)
 [S] » [S]initial

ASSUMPTION #5:
 The enzyme is either present as free enzyme or as the ES complex
 [E]total = [E] + [ES]
 Michealis-Menten Analysis
• Michaelis–Menten kinetics is one of the simplest and best-
known models of enzyme kinetics.

• The model serves to explain how an enzyme can cause

kinetic rate enhancement of a reaction and why the rate of a
reaction depends on the concentration of enzyme present.

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To begin our discussion of enzyme kinetics, lets define the
number of moles of product (P) formed per time as V.

• The variable, V, is also referred to as the rate of catalysis of

an enzyme.

• For different enzymes, V varies with the concentration of the

substrate, S.

• At low S, V is linearly proportional to S, but when S is high

relative to the amount of total enzyme, V is independent of S.

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To understand Michaelis-Menten Kinetics, we will use the
general enzyme reaction scheme shown below, which includes
the back reactions in addition the forward reactions

[ ] --1

Rate Reaction Constant

K1: The binding of the enzyme to the substrate forming the
enzyme substrate complex.
K-1: The dissociation of the enzyme-substrate complex to free
enzyme and substrate .
K2: Catalytic rate; the catalysis reaction producing the final
reaction product and regenerating the free enzyme. This is
the rate limiting step.

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 So in the Steady State,

1. Formation of ES : k1 is constant
Rate of formation of [ES ] = k1 [E][S]
2.Rate of dissociation / breakdown of [ES]
Two Types a. (E S) to E +S ; k-1 is constant
a. Rate of disassociation of E S = k-1( ES )
b. (E S) to E + P ; k2 is constant
b. Rate of disassociation of (E S) = k2 (ES)
Rate of dissociation of [ES] = k-1 [ES] + k2 [ ES]
Rate of formation of [ES ] = k1 [E][S]
Rate of dissociation of [ES] = k-1 [ES] + k2 [ ES]
Rate of formation of [ES ] = Rate of dissociation of [ES]
k1 [E][S] = k-1 [ES] + k2 [ ES]
Factor out ES
= [ES] (k-1 + k2)
k1 [E][S] = [ES] (k-1 + k2)
[E][S] (k-1 + k2)
-------------- = -------------- Rate constants
[ES] k1
Ratio of Rate constants is defined as Michaelis constant
and is abbreviated as Km
[E][S] = Km
--------------
(ES]
(E][S]
Km = -------------
(ES]
 [ES] = [Eo]-[ES] [S]/ Km

[Eo] [S] – [ES] [S] = Km [ES]

[Eo] [S] = Km [ES]+ [ES] [S]

Take out [ES]
[Eo] [S] = ES ( Km + S)

[Eo] [S]
--------------- = ES This is converted into Product P + E
( Km + S)
This is the Steady State condition and Assumption No: 1
Velocity Vo is determined by the breakdown of ES to form
product which is determined by [ES]
V = k2[ ES]
Or
Vo= dP/dt = k2[ ES]

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 Km = [E][S]
------------- OR Km= [E] [S]/[ES]
[ES]
Since the enzyme is not consumed, the conservation equation on the
enzyme yields : [ET] = [ES] + E
[E] = [ET]-[ES]
So putting the value of [E] in to the equation : Km= [E] [S]/[ES]

Km= [ET]-[ES] [S]/[ES]

Changing/ Cross [ES] and Km sides
[ES] = [ET]-[ES] [S]/ Km
So finally,
[ES]= [ET][S] / Km +[S]
Then putting the value of [ES] from the above equation in equation
no: Vo= k2[ES], the rate V0 can be expressed in terms of [S].
V0 = k2 [ET][S] / Km +[S]
Vmax= k2 [Eo]
Finally,
Vo = Vmax [S] / Km +[S]

The equation -8 is called the Michaelis-Menten equation, for a one-

substrate enzyme-catalyzed reaction.

Note # when V0 is exactly one-half Vmax:

V0/2 = Vmax [S] / Km +[S]

Maximal Velocity (Vmax): Increasing the substrate concentration
indefinitely does not increase the rate of an enzyme-catalyzed reaction
beyond a certain point. This point is reached when there are enough
substrate molecules to completely fill (saturate) the enzyme's active sites.
The maximal velocity, or Vmax, is the rate of the reaction under these
conditions. Vmax reflects how fast the enzyme can catalyze the reaction.
Click on the image at right to see how high Vmax and low Vmax enzymes
compare. Vmax is given by the asymptote to the velocity curve as the
substrate concentration is extrapolated to infinity. Notice that Km stays
constant for the two enzymes described here.