ENZYME KINETICS

COVERAGE
BASIC ENZYME REACTION

MICHAELIS-MENTEN KINETICS

MULTISUBSTRATE SYSTEMS

ENZYME INHIBITION

PH AND TEMPERATURE EFFECTS

 ENZYME KINETICS
The study of the chemical reactions
that are catalyzed by enzymes

Catalytic mechanism
Role on metabolism
How activity is controlled
How might inhibit the enzyme
 BASIC ENZYME REACTION
ENZYME-SUBSTRATE
FREE ENZYME
COMPLEX

E +S ES E+P

PRODUCT
SUBSTRATE
 Proposed and
postulated a
mechanism
dependence of the
initial rate of enzyme-
catalyzed reactions on
concentration.
LEONOR MICHAELIS MAUD MENTEN

E+S ES ES E+P
MICHAELIS-MENTEN
KINETICS
 1
E+S 1
ES 2 E+P

=
+

Michaelis-Menten Equation
 k--1k2
[]
= =
[]
[] ( ) []
=

= [] =
[]

[] = + []
[]
[] =
+ []
[] []
= = [S]
KS

+ []
[]
[]
== []
== []

= 2 []

DERIVATION Maximum Rate

 If, [S] = KS.

=
2
STEADY-STATE KINETICS
Known as the Briggs-Haldane Assumption

There is no need to assume that enzyme substrate are in

thermodynamic equilibrium with the enzyme-substrate
complex to derive the equation of initial velocity.

The concentration of the enzyme-substrate

complex will reach a constant value so that we
can apply the steady-state approximation
soon after enzyme and substrate combined
[] [] []
= = = =

( + )[] + +

[] [] []
= = =
+ +
[] []
=
[ + ] + []

=
+
+
=
DERIVATION
 INTERPRETING Vmax AND KM

Two of the important parameters on Michaelis-Menten

KM equation can be experimentally obtained for any given enzyme
Vmax

Double-Reciprocal Plot
A more convenient way of obtaining
these two parameters without the
use of simple graphical method
 Michaelis-
Menten Equation
[] []
= = = +
+ + [] [] []

= +
[]

Lineweaver-
Burk Equation
 THE SIGNIFICANCE Vmax AND KM

pH The Michaelis constant KM

and the maximum rate,
Vmax can be readily derived
Temperature from rates of catalysis
measured at a variety of
Ionic Strength substrate concentrations if
an enzyme operates
according to simple
Particular Substrate scheme given in the
equation

Factors Affecting KM
 KM is the concentration of the substrate at
KM which half the active sites are filled

KM gives a measure of the

substrate concentration required
for significant catalysis to occur

[]
= =
+
A while ago Under such circumstances, the
+
ES complex dissociates to E and
=

S much more rapidly than =
product is formed. Under these
conditions (k-1 k2),

[]
= = The dissociation constant of the ES complex
[]

KM is equal to the dissociation constant of the

ES complex of k2 is much smaller than k-1 KM
KM Binding
Vmax The turnover number of an enzyme, which the
number of substrate molecules converted into
product by an enzyme molecule in a unit time
when the enzyme is fully substrate

Turnover Number

= [] = /[]
 KINETIC PERFECTION IN ENZYMATIC CATALYSIS:
THE kcat/KM CRITERION

When the substrate concentration

is much greater than KM, the rate
of catalysis is equal to kcat, the
turnover number, as described
in previously

Under physiological conditions, the [S]/KM ratio is typically

between 0.01 and 1.0. When [S] <<KM, the enzymatic rate is
much less than kcat because most of the active sites are unoccupied
 [S] << KM

= [] = [][]

Thus, when [S] << KM,
the enzymatic velocity
This rate cannot be faster than the diffusion- depends on the values
controlled encounter of an enzyme and its ofkcat/KM, [S], and [E]T.
substrate. Diffusion limits the value of k1 so
that it cannot be higher than between
108 and 109 s-1 M-1. Hence, the upper limit Catalytic Efficiency
on kcat/KM is between 108 and 109 s-1 M-1.

= = < 1
1 + 1 + 1 1
 Enzymes such as these that have
kcat/KM ratios at the upper limits have
attained kinetic perfection. Their
catalytic velocity is restricted only by Any further gain in catalytic rate
the rate at which they encounter can come only by decreasing the
substrate in the solution time for diffusion
 MULTISUBSTRATE SYSTEMS
Bisubstrate
Reaction

SUBSTRATES
A+B P+Q PRODUCTS

The binding of A and B to

the enzyme can take place
in different ways
SEQUENTIAL NONSEQUENTIAL
MECHANISM MECHANISM
 SEQUENTIAL MECHANISM
ORDER SEQUENTIAL RANDOM SEQUENTIAL

In this mechanism, . The case which binding

of substrates and the
one substrate release of products do
must bind before not follow a definite
obligatory order known
second substrate as a random sequential
can bind mechanism.
 ORDER
SEQUENTIAL
A B P Q

E EA EAB EPQ EQ E

RANDOM
A B P Q SEQUENTIAL
EA EQ

E EAB EPQ E
EB EP
B A P Q
NONSEQUENTIAL MECHANISM
PING-PONG MECHANISM

In this mechanism, one substrate binds, and one product is

released. Then, a second substrate binds, and a second
product is released.

A P B Q

E EA E* E*B E
 ENZYME INHIBITION
Inhibitions are compounds that decrease
the rate of enzyme-catalyzed reaction.

A B C D E F
 REVERSIBLE In reversible inhibition, an
INHIBITION equilibrium exists between
the enzyme and the inhibitor.

ACTION OF
INHIBITOR
In irreversible inhibitions,
IRREVERSIBLE inhibition progressively
INHIBITION increases with time
 COMPETITIVE

REVERSIBLE
NONCOMPETITIVE
INHIBITIONS

UNCOMPETITIVE
 COMPETITIVE INHIBITION

E+S ES P+E Both the substrate S

and the inhibitor I compete
+ for the same active site
I EI
 [] []
=
+
[]
+ []
= + +
[]
Applying the steady- the Lineweaver-
state approximation Burk equation for
for ES this is given by
 NONCOMPETITIVE INHIBITION
binds to the enzyme at
E+S ES P+E a site that is distinct from the
+ substrate binding site; thus, it
can bind to both the free
I I enzyme and the enzyme-
substrate complex
EI + S ESI

[]
[] [] []
+
= + + +
= []
+ []
Applying the steady- the Lineweaver-
state approximation Burk equation for
for ES this is given by
 UNCOMPETITIVE INHIBITION

Does not bind to the free

E+S ES P+E enzyme; Instead it binds
+ reversibly to the enzyme-
I substrate complex to yield in an
inactive ESI complex

ESI

[]
[]
+
=
[]

+ [] = + +
+
[] []

the Lineweaver-
Applying the steady- Burk equation for
state approximation this is given by
for ES
 Michaelis-Menten kinetics
cannot be applied to
irreversible inhibition. The
inhibitor forms a covalent
IRREVERSIBLE linkage with the enzyme
INHIBITIONS molecule and cannot be
removed.
 EFFECT OF pH ON ENZYME
The most favorable pH value - the point where the enzyme
is most active
OPTIMUM pH

pH is also a
factor in the
stability of
enzymes
 EFFECT OF TEMPERATURE ON ENZYME

Like most chemical reactions, the rate of an enzyme-catalyzed reaction

increases as the temperature is raised. A ten degree Centigrade rise in
temperature will increase the activity of most enzymes by 50 to 100%

In the case of
enzymatic reactions,
this is complicated by
the fact that many
enzymes are
adversely affected by
high temperatures.
 In addition to
temperature and pH
there are other
factors, such as ionic
strength, which can
affect the enzymatic
reaction