Enzyme Kinetics

Rate of Enzyme Catalysis vs Substrate

Concentration
For many enzymes, the rate of
catalysis, V, varies with the
substrate concentration, [S].
V is defined as the number of
moles of product formed per
second.
At a fixed concentration of
enzyme, V is almost linearly
proportional to the initial
increasing concentration of [S].
At very high [S], V is
independent of [S].

The Michaelis-Menten Approach to Enzyme

Kinetics
(Leonor) Michaelis and (Maud) Menten devised a model for the kinetics of
enzyme-catalyzed reactions.

The model proposed is the one that accounts for the kinetic properties of many
enzymes:

An enzyme, E, combines with S to form an ES complex, with a rate constant k1.

The ES complex has two possible fates. It can dissociate to E and S, with a rate
constant of k-1, or it can proceed to form product, P, with a rate constant k2.
Michaelis and Menten made a derivation of these kinetic characteristics to arrive
at the Michaelis-Menten Equation:

Vinit =

Vmax [S]
KM + [S]

Michaelis-Menten
equation

Michaelis-Menten Equation Derivation

For an enzyme-catalyzed reaction
E + S

k1
k-1

ES

k2

The rates of formation and breakdown of ES are

given by these equations
rate of formation of ES = k
rate of breakdown of ES = k

1 [E][S]
-1 [ES]

At the steady state

k1 [E][S] = k-1[ES] + k 2[ES]

+ k2[ES]

Michaelis-Menten Equation Derivation (cont.)

When the steady state is reached, the concentration
of free enzyme is the total less that bound in ES
[E] = [E]T - [ES]

Substituting for the concentration of free enzyme and

collecting all rate constants in one term gives
([E]T - [ES]) [S]
[ES]

k-1 + k2

= KM

k1

Where KM is called the Michaelis constant

Michaelis-Menten Equation Derivation (cont.)

It is now possible to solve for the concentration of the
enzyme-substrate complex, [ES]
[E]T [S] - [ES][S]
[ES]

= KM

[E]T [S] - [ES][S] = KM [ES]

[E]T [S] = [ES](K M + [S])

Or alternatively

[ES]

[E]T [S]
=
KM + [S]

Michaelis-Menten Equation Derivation (cont.)

In the initial stages, formation of product depends only on the
rate of breakdown of ES

Vinit

= k2 [ES]

k 2[E]T [S]
=
KM + [S]

If substrate concentration is so large that the enzyme is

saturated with substrate [ES] = [E]T

Vinit

= Vmax = k2 [E]T

Substituting k2[E]T = Vmax into the top equation gives

Vinit =

Vmax [S]
KM + [S]

Michaelis-Menten
equation

The Meaning of KM or the Michaelis constant

Initial concentration of substrate at Vmax or at half
saturation of E with S
Ratio of the constants for the breakdown of ES to
constants of its formation:
[E]T [S] - [ES][S]
[ES]

= KM

[E]affinity
T [S] - [ES][S]
A measure of the
of E for= S:KifM [ES]
k2 is <k-1 or k1,
neglecting k2, therefore [E]
KM =[S]
k-1=/k[ES](K
1
T
M + [S])
The lower the KM value, the more affinity has the
enzyme for its substrate

The Meaning of KM or the Michaelis constant

When [S]= KM, the equation reduces to

V=

Vmax [S]
KM + [S]

Vmax [S]
[S] + [S]

Vmax
2

Linearizing The Michaelis-Menten Equation

o It is difficult to determine Vmax experimentally
o The equation of Michaelis and Menten is for a hyperbola

V=

Vmax [S]
KM + [S]

(an equation for a hyperbola)

o The equation can be transformed into the equation for a

straight line by taking the reciprocal of each side

1
V
1
V

KM + [S]

Vmax [S]
KM
Vmax [S]

KM
Vmax [S]
1

Vmax

[S]
Vmax [S]

Lineweaver-Burk Plot
The Lineweaver-Burke plot has the form y = mx + b, and is
the formula for a straight line

1
V

KM
=
Vmax

[S]

1
Vmax

a plot of 1/V versus 1/[S] will give a straight line with slope
of KM/Vmax and y intercept of 1/Vmax
such a plot is known as a Lineweaver-Burk double
reciprocal plot
Double reciprocal plots can easily be understood and
provide recognizable pattern for the study of ENZYME
INHIBITION

Lineweaver-Burk Plot (Contd)

o KM is the
dissociation
constant for ES;
the greater the
value of KM, the
less tightly S is
bound to E
o Vmax is the
turnover
number

Turnover Numbers
Vmax is related to the turnover number of enzyme:also
called kcat

V max

turnover _ number kcat

[ET ]
o Number of moles of substrate that react to form
product per mole of enzyme per unit of time

Enzyme Inhibition
Reversible inhibitor: a substance that binds to an enzyme to inhibit
it, but can be released

competitive inhibitor: binds to the active (catalytic) site

and blocks access to it by substrate
noncompetitive inhibitor: binds to a site other than the
active site; inhibits the enzyme by changing its
conformation

Irreversible inhibitor: a substance that causes inhibition that cannot

be reversed

usually involves formation or breaking of covalent bonds

to or on the enzyme

Competitive Inhibition
o Substrate must compete with inhibitor for the active
site; more substrate is required to reach a given
reaction velocity
EI

E + S

ES

o We can write a dissociation constant, KI for EI

EI

KI =

[E][I]
[EI]

Competitive Inhibition

Competitive Inhibition
No inhibition
1 = KM
V
Vmax
m

y =

1
S

1
Vmax
b

In the presence of a competitive inhibitor

1
V

KM
Vmax

1 +
m

[I]

KI

1
S

1
Vmax
b

In a Lineweaver-Burk double reciprocal plot of 1/V

versus 1/[S], the slope (and the x intercept) changes but
the y intercept does not change

A Lineweaver-Burke Plot for Competitive Inhibition

Noncompetitive Inhibition (Contd)

Several equilibria are involved
+S

E
-I

ES

E + P

-S
+I

-I
+S

EI

+I

ESI

-S

The maximum velocity Vmax has the form

max =

Vmax
1 + [I]/K I

Noncompetitive Inhibition (Contd)

A Lineweaver-Burke Plot for Noncompetitive Inhibition

o Because the inhibitor does not interfere with binding of
substrate to the active site, KM is unchanged
o Increasing substrate concentration cannot overcome
noncompetitive inhibition
No inhibition
1 = KM 1
V
S
Vmax

1
Vmax

y =
m x + b
In the presence of a noncompetitive inhibitor

1
V

KM
Vmax

1 +
m

[I]

KI

1
S

1
Vmax

1 +

[I]
KI

A Lineweaver-Burke Plot for Noncompetitive Inhibition

(Contd)

Other Types of Inhibition

Uncompetitive- inhibitor can bind to the ES complex but
not to free E. Vmax decreases and KM decreases.

Mixed- Similar to noncompetitively, but binding of I

affects binding of S and vice versa.

Uncompetitive Inhibition

Uncompetitive Inhibition

Summary
Effects of reversible inhibitors on kinetic constants
Type of Inhibitor

Effect

Competitive (I binds to E
only)

Raises KM; Vmax

unchanged

Uncompetitive (I binds to
ES only)

Lowers KM and Vmax

Non-competitive (I binds to
E or to ES)

Lowers Vmax; KM
unchanged