NPTEL – Biotechnology – Systems Biology

Michaelis Menten Kinetics-1

Dr. M. Vijayalakshmi
School of Chemical and Biotechnology
SASTRA University

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Table of Contents
1 INTRODUCTION .............................................................................................. 3
1.1 SINGLE SUBSTRATE REACTIONS ..................................................................... 3
1.1.1 Assumptions in Michaelis and Menten Kinetics ..................................... 3
1.2 SIGNIFICANCE OF MM CONSTANTS .................................................................. 6
2 REFERENCE .................................................................................................... 9
2.1 TEXT BOOKS ................................................................................................. 9

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1 Introduction
In the previous lecture, we discussed the importance and basics of enzymatic reactions in
biological systems. Let us now understand how one can interpret the kinetics of an
enzyme catalysed reaction. To understand the mechanism of any catalytic reaction, one
should study the kinetic behaviour of the reaction systems, where the rate of the reaction
can be obtained at various concentrations of the enzyme and the substrate.

Formation of enzyme substrate complex is a critical event in an enzyme-catalyzed

reaction. Michaelis and Menten proposed that the binding of the substrate and the enzyme
is reversible and derived a kinetic model for a simple single substrate enzymatic reaction.
This laid the basis for understanding the various factors that influence rate of the reaction,
such as concentration, temperature and pH of the system.

1.1 Single Substrate Reactions

Consider a simple single substrate reaction, where the free enzyme E binds to the
substrate S to form a complex ES, the forms product P and then dissociates.

The dissociation of ES complex into free enzyme and product is the slowest and hence it
is the rate-limiting step in the reaction. Throughout the reaction, the total concentration of
the enzyme will be the sum of concentration of total free enzyme [E] and concentration of
total enzyme bound with substrate [ES].

At pre-steady state, substrate concentration is more than that of the enzyme

concentration. As reaction proceeds, the concentration of the enzyme substrate complex
increases with time and reaches a steady state. After this stage, the concentration does
not change further with time.

1.1.1 Assumptions in Michaelis and Menten Kinetics

a) Once the system attains a steady state, the concentration of ES remains the same
throughout the process.
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b) Initially, there is no product formation and hence the reverse conversion of the
product to the substrate cannot happen. Hence K2 can be neglected.

The free enzyme [E] at any point of time in the reaction will be equal to the concentration
of the enzyme substrate complex [ES] subtracted from the total enzyme concentration
[ET].
[E] = [ET] – [ES]

Rate of formation of ES = K1 ([ET] – [ES]) [S]

Rate of breakdown of ES = K-1 ([ES] + K2 [ES]

From the steady state assumption,

rate of formation = rate of breakdown

K1 ([ET] – [ES]) [S] = K-1 ([ES] + K2 [ES]

K1 ([ET] [S] – K1 [ES]) [S] = (K-1 + K2 ) [ES]
K1 ([ET] [S] = [ES] (K-1 + K2 + K1[S] )

[ES] = (K-1 + K2 + K1[S] )

Here, Michaelis-Menten rate constant is defined as,

The equation can be simplified as:

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Rewriting the rate for breakdown of ES, we can represent ES as V 0 in the above equation
to get,
V0 = K2 [ES]
[ES]= V0 / K2
Substituting [ES] as V0 gives

Maximum velocity can be obtained only when the enzyme is completely saturated. At this
point, the total enzyme concentration will be equal to the concentration of enzyme
substrate complex alone.

Changing [ES]= [ET] and V0 = Vmax , Vmax = K2 [ET]

Therefore the expression for V0 can be rewritten as,

This is the MM equation for a single substrate enzyme catalysis reaction. Michaelis
Menten kinetics also gives the numerical interpretation that V0 is half the Vmax.
When

The MM kinetic expression forms a rectangular hyperbola and the graph is given in Fig 2
below

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Fig 1: Concentration profile of enzyme, substrate, ES and product with respect to time

Fig 2: A typical plot of the Michelis Menten kinetics

1.2 Significance of MM constants

Km: Km is a strong experimental parameter, which provides information on affinity

shown by substrates towards the enzyme.

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For a single substrate reaction, the breakdown of ES into E and S is the slowest step and
hence k-1 >> k2 => Km = k-1/k1.
At this condition, Km gives a measure of affinity between substrate and enzyme and can
be used as dissociation constant.

When k-1 >> k2 or k-1= k2, the expression of Km will become complex and cannot directly
give an overview of the affinity between substrate and enzyme.

The value of Km for any enzyme is influenced by parameters such as pH and temperature.
The fraction of enzyme substrate complex can be expressed in terms of Km as:

Vmax
Vmax gives measure of catalytic efficiency of a particular enzyme over its substrates.

K2
K2 is defined for the rate limiting steps of the enzymes with multiple catalytic steps (sec-1).
When

It represents the number of molecules of substrate converted into product at a unit of time
by a single enzyme molecule and is termed turnover number (Kcat). This is used to
compare the catalytic efficiencies of different enzyme groups.
When [S] >> Km,
Rate of catalysis will be Kcat and enzymatic velocity depends on Kcat/Km and ET. The term
Kcat/Km, serves as the measure of catalytic efficiency of the enzyme.

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Generally, the upper limit for the kcat /KM is between 108 and 109 s-1 M-1. Enzymes having
Kcat /Km values in the upper limits are said to have attained kinetic perfection and their
catalytic efficiency is restricted only by the substrate concentration in the solution.
To ease the experimental determination of these Michaelis-Menten kinetic constants,
different plots have been arrived at. These are discussed in the next section.

Lineweaver-Burk (double reciprocal plot)

By taking reciprocal of the MM equation, we arrive at a relation that resembles the

equation of the straight line y = mx + b

Here,
y = 1/Vo
m (slope) = Km/Vmax
x = 1/[S]
y- intercept, b = 1/Vmax

This plot helps in accurate determination of Vmax and Km, classification of different
mechanisms of action of enzymes and the effect of the inhibitors. Other plots like Hanes –
Wolf plot are also used to calculate the Michaelis-Menten kinetics of an enzymatic
reaction. So far, we have understood how the kinetics of single substrate enzymatic

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reaction is interpreted. In the next lecture, we shall discuss how the enzyme inhibition
happens with the structural analogs of the substrate molecules.

2 Reference

2.1 Text Books

1. Bisswanger H, Enzyme Kinetics, Principles and Methods, WILEY-VCH

(2002).
2. J. D. Murray, Mathematical Biology, Springer-Verlag, (1989).
3. Berg JM, Tymoczko JL, Stryer L. Biochemistry, 5/e, W H Freeman, (2002).

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