Last Update: 2 November 2017 Part I

ENZYME KINETICS M-9

Kinetic Properties of Enzymes and Derivation of Michaelis Menten equation

Enzymes are extraordinary bio-catalyst. All the enzymes are protein except ribozymes. Enzyme
increases the reaction rate of about 7 to 14 orders. They are also very specific for their particular substrate.
During formation of enzyme substrate complex a small amount of free energy release that lower the
activation energy of reaction.

Ribozyme

A ribozyme (from ribonucleic acid enzyme, also called RNA enzyme or catalytic RNA) is an RNA molecule that
catalyzes a chemical reaction. Many natural ribozymes catalyze either their own cleavage or the cleavage of other
RNAs, but they have also been found to catalyze the aminotransferase activity of the ribosome. Investigators studying
the origin of life have produced ribozymes in the laboratory that are capable of catalyzing their own synthesis under
very specific conditions, such as an RNA polymerase ribozyme. More work needs to be done in this area though, as
the polymerase ribozyme does not have enough catalytic prowess: it is able to add up to 14 nucleotides to a primer
template in 24 hours until it is decomposed by hydrolysis of the phosphodiester bonds

Example: A recent test-tube study of prion folding suggests that an RNA may catalyze the pathological protein
conformation in the manner of a chaperone enzyme.Some known ribozymes include RNase P, Group I and Group II
introns, leadzyme, hairpin ribozyme, hammerhead ribozyme, hepatitis delta virus ribozyme, and tetrahymena
ribozyme.

Kinetics of enzymatic reaction starts with the substrate concentration [S] and initial velocity V o.
When substrate concentration is very low, initial velocity also low, if substrate concentration increases
progressively but enzyme concentration is constant then Vo increases almost linearly with as increasing
substrate concentration [S]. At very high substrate concentration Vo increases by smaller and smaller
amounts and finally reach a plateau that is called maximum velocity i.e. Vmax
Enzyme-substrate (ES) complex is the key to understanding the kinetic behaviors of enzymatic
reaction that is proposed by Victor Henri 1903. This idea
expended and give a general theory of an enzymatic reaction
by Leonor Michaelis and Maud Menten in 1913.
Fig. 1. shows the relationship between [S] and V0 for
an enzymatic reaction. The hyperbolic shape of this curve
can be expressed algebraically by the Michaelis-Menten
equation. Derivation of Michaelis-Menten equation stars
with two basic reaction involved in the formation and
breakdown of ES.
K K
E + S 1 ES 2 E + P
K 1 K 2
In equation , E = Enzyme.
P = product, S = substrate, ES = Enzyme substrate complex, K1 = rate of constant of ES
formation, K2 and K -1 are the rate of constant of ES break down. However K -2 is negligible
and ignored.
V0 is initial velocity determine from the break down of ES.
V0 = K2 [ES](2)

-1-
 Formation of [ES] can not measure easily in experimental condition, so we can use the term total
enzyme [Et]. From [Et] we can calculate free or unbound enzyme that is [Et]-[ES]. So the comparison
between [S] and [Et] is negligible.
Rate of ES formation = K1 ([Et] - [ES]) [S]
Rate of ES break down = K1 [ES] + K2 [ES]

During the equilibrium condition, the initial rate of reaction reflects a steady state in which ES is constant
and rate of [ES] formation is equal to rate of [ES] break down.
At steady state
K1 E t ES S K 1ES K 2 ES

or, K1S E t k1S ES K 1 ES K 2 ES

or, K1S E t k1 S ES K 1ES K 2 ES

or, K1S E t ES K1 S K 1 K 2

K1 E t S
or, ES
K1 S K 1 K 2

or, ES
E t S
S K 1 K 2
K1

or, ES
E t S the term (K + K ) / K is define as the Michaelis Menten constant, K
Km S
2 1 1 m.

K 2 E t S
or, V0 Vo can now be expressed in term of ES (from Eq..2)
K m S

The maximum velocity Vmax is reached when enzyme is saturated by substrate then, [ES] = [Et].
From equation . (2) we get
Vo = K2 [ES]
Vo = K2 [Et]
Then Vmax = K2 [Et]

Vmax S
Vo
K m S
(Michaelis-Menten equation)

Km is the Michaelis-Men ten constant define as molar concentration of substrata when Vo reaches
1
exactly Vmax.
2
1 Vmax S
Then Vmax =
2 K m S
Km = [S]
So, Km is equivalent to Substrate concentration of which V0 = V max and unit of Km is mole / liter.

-2-
Significance of Km
i) Km indicates the degree of affinity of an enzyme for its substrate. Substrate affinity is inversely proportional
with Km value.
1
K m
Substrate afinity
The higher Km value indicates lower substrate affinity or the lower Km value indicates higher substrate
affinity

ii) The range of Km value is about 10 6 to 10 1 mole / 1 litre

iii) Km is not influenced by enzyme. Concentration or non-competitive inhibition but it is changed by

competitive inhibition (increase) or uncompetitive inhibition (decrease)
iv) Temperature, alosteric modulations ionic concentration alters the Km value.
v) Km value of an isoenzyme is different.

1) Linier transformation of Michaelis-Menten equation

The double reciprocal plot or Line weaver Burk
plot.
Michaelis- Menten equation :

Vmax S
Vo
K m S

1 K m S
or,
Vo Vmax S

1 K 1 1
or, m
Vo V max [S] V max

1 1
Plotting of against gives a straight Line that
Vo S
is double reciprocal plot or Line Weaver Burk plot.

[S]
2) Wolf - Hanes Plot : Plotting of against
Vo
respective values of [S] gives a straight line called
Wolf- Hanes plot

The derivation is
Vmax S
Vo
K m S

1 K m S
or,
Vo Vmax S

or,
S K m S
Vo Vmax

-3-
or,
S Km
S
1
Vo Vmax Vmax

3) Eadie - Hofstee plot :-

Vo
Plotting against Vo and gives an another linear form of Michaelis-Menten equation that is called Eadie
[S]
- Hofstee plot
Vmax S
V0
K m S

Vo Vmax
or,
S K m S
Vo
or, K V V
S m o max
Vo
or, Vo Vmax K
S m

Q: a)Write the Michaelis-Menten equation for a single substrate reaction.

b) What is the form of Michaelis-Menten equation under the following conditions :
i) when [S] = Km;
ii) when [S] >> Km;
iii) when [S] << Km.

Following is the Michaelis-Menten equation for a single-substrate enzymatic reaction obeying the
hyperbolic saturation kinetics:

Vmax S Vmax
V ; K m [ S ] 1 (Michaelis-Menten equation) Where
K m S
KM is called the Michaelis
V
constant.
When V = Vmax only at that condition [S] will equal to Km, as it is derived putting the value of Vmax at
the position of V in the above Michaelis-Menten equation and gives
V
K m [ S ] 1 max 1 = [S]
2 Vmax

So, Km is the molar concentration of the substrate at which half the maximal velocity is attained. Km
ranges from about 10-6 to about 10-1 mole per litre. It indicates the degree of affinity of an enzyme for its
substrate. The lower the Km value, the higher is the substrate affinity of the enzyme. The substrate affinity
of an enzyme and consequently its Km varies from substrate to substrate.

-4-
 The initial velocity V is directly proportional to the molar concentration [S] of the substrate when the
substrate concentration is very low compared to the Km ([S] << Km). In this state, a single-substrate enzymatic
reaction is a first order reaction, its rate depending on the concentration of the single reactant.
[S] << Km, Km + [S] Km

Vmax S Vmax S
V
K m S
= K[S]
Km
Where K is a new constant equalling because both Vmax and Km are constants for a particular enzyme.
On the contrary, when [S] far exceeds Km ([S] >> Km), the initial velocity attains its maximal value Vmax and
becomes independent of [S]. The reaction now turns into a zero order reaction, its rate independent of the
reactant concentration.
[S] >> Km, Km + [S] [S]

Vmax S Vmax S
V
K m S
= K[S]
Km

-5-