ENZYME KINETICS

Michael-Menten Approach

Enzymatic reaction

Simplest enzymatic reaction 1st order
(1)

Real enzyme mechanism  more complexes;

never proceed through just 1 ES complex.

Involved many steps pathway

The slowest one determines the rate of overall
reaction

How the reaction rate is affected by reaction
conditions (S,P,E) important, to understand the
effectiveness & characteristics of enzyme reaction

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The reaction will start at a high
rate and slow down over time
for several reasons:

The substrate will be used up
and the reaction rate will slow
down as each enzyme
molecule spends more time
diffusing through the solution
before it collides with a new
substrate molecule.

As the reaction proceeds,
product will accumulate which
may tend to inhibit the
enzyme.

The enzyme molecules may
gradually lose activity owing
to random denaturation.

Graph
Plotting r0 at different [S] &
[E]:

r is proportional to [S]
(1st-order reaction) 
when the [S] is in the low
range

[S] very low  a straight line
graph

when [S] is low, [S]  the
limiting factor; an increase [S]
produces a proportional
increase in the rate

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graph will curve at higher
the enzyme becomes
[S] & will level off at very "saturated" and the
high [S] reaction rate reaches a

The rate  does not constant value doesn't
depend on the [S] when increase significantly as
the [S] is high yet more substrate is

Reaction rate changes added.
gradually from first order
to zero order as [S] is
increased.

[S] increases, [E]  the
limiting factor

a further increase in
substrate  produces a
less than-proportional
increase in reaction rate.

ENZYME KINETICS

deals with the rate of enzyme reaction

Rate (r), activity, dP/dt, (-dS/dt) how fast an
enzyme catalyses S to P, the amount of S
consumed, or P formed per unit time

Rate ?  need S or P, & t

how reaction is affected by various chemical and
physical conditions

The rate equations  calculating reaction time,
yields

For designing bioreactor and optimum economic
condition

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Factors affecting the rate of enzyme
reaction

[S]
the more, the quicker the enzyme molecules collide and
bind with them). expressed in the unit of “molarity”, M

T
As the T rises, molecular motion speed up collisions
between enzyme and substrate. Finite  enzymes are
proteins, have an upper limit beyond which the enzyme
becomes denatured and ineffective.

inhibitors
a. Competitive inhibitors
b. Noncompetitive inhibitors

pH
The conformation of a protein is influenced by pH and as
enzyme activity is crucially dependent on its conformation,
its activity is likewise affected.

Rate vs [S]
• [S] low  they are all bound to
the enzyme molecules
• there are more substrate
molecules some of them are
free in solution, not bound to an
enzyme molecule.
• [S] high  all the enzyme
molecules have a bound
substrate.
• The number of enzyme molecules
with bound substrate is an
indication of the reaction rate 
those enzymes "act" and convert
the substrate to product.
• The purple highlight indicates the
region of the graph that
corresponds to illustration in the
left.

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Henry (1902) observed Assumptions:
this & proposed: 1. The total enzyme
(2) concentration  constant
during the reaction; [Eo] =
[ES]+[E]
rmax & Km  kinetic
parameters, 2. The enzyme is very small
experimentally determined amount compared to the

Brown (1902) “E forms a amount of substrate  the
complex with S. The formation of the complex
complex then breaks down does not significantly deplete
to the products, the substrate.
regenerates free enzymes
(3) 3. [P] is so low that product
inhibition may be considered
(4) negligible

MICHAEL-MENTEN APPROACH
Assumption for MM approach:

most enzymes  has a fairly simple math shape
a hyperbola

product release step (4) is much slower than
reversible reaction (3)

ES complex has a weak interaction fast
reaction; much faster step than product release
step which involved chemical changes

the slowest step determine the rate, the other is
at equilibrium

eventhough enzyme is soluble in water, enzyme
molecules have large & complicated 3D structure

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If slower reaction (4) determines overall reaction rate of
P formation & S consumption is proportional to CES:
(5)

Assumption MM no.3  CES =f(Cs, CE):

(6)

the rate of reaction can be expressed as a function of Cs

and CE, CE difficult to be determined

Total enzyme contents:
(7)

Substitution (6) to (5) for CE & rearrange CES:

(8)

Final rate eq:

(9)

Know as MM eq identical to (2)

Assumption: 1) only one intermediate state (ES complex), 2)
dealing with initial rate (not necessary to consider back reaction)

Km, Cs  same unit (M or mol/L)

Rmax is proportional to the CE0  rmax=k3CE0;difficulty to
express CE0 in M unit

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From MM eq

Initial [S] increase reaction
[S]=Km rate=one-half of
goes faster; enzymes easier rmax
to find S

[S]<<Km rate depends

All active sites occupied with linearly on [S]; 2x[S]  2x
bound S or P  saturated
rate

[S]<<<[Km]  double const,
double rate
[S]>>Km dependence of

rmax  rate approached at rate on [S] approach a
saturated [S]; rate does not max horizontal
depend on [S] line/independent; rmax

Km: [S] required to produce zero order kinetic
rate that is one-half of rmax

Km >> the weaker the
interaction between enzyme &
S

Theory shows that KM is approximately equal to the

dissociation constant for the enzyme and the substrate,
provided that the enzyme-substrate complex reverts to
enzyme and substrate much more often than it goes on to
generate product

Enzymes have higher rates of reaction  the substrate
concentration is greater than KM.

All enzyme-catalyzed reactions are reversible and if the
concentration of product is high, the reverse reaction will
compete with the forward reaction for enzyme.

The rmax and KM values for the reverse reaction are
usually different from those of the forward reaction.

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 Alternate Forms of the MM eq.

Where does KM & rmax
if KM is high, the curve will
come in? appear to spread out toward

Estimate Km & rmax  the right, approaching Vmax
asymptote only at high substrate
concentration.

Km=f ([S])
Km = rmax/2

Km low  corresponds to
tight binding of enzyme to
substrate and vice versa

KM is large the rate at
low [S] is relatively low.

KM is small the rate at
low [S] is relatively high

The curve of r vs [S]  rise
quickly if KM is low and the
curve will soon approach
Vmax

rmax  a property of the
Km, depends on the

enzyme, interaction of E &
S substrate being used and

The rmax of an enzyme is
a  how fast the reaction
it catalyzes can proceed
once the enzyme-substrate
complex is formed

rmax =f ([E, T, pH ionic
strength in solution])

rmax  to the turnover
number of substrate
molecules converted into P
per active site at very high
substrate concentration.
particular enzyme and
on the conditions of
temperature, pH, and ionic
strength in the solution

Km is independent of the
enzyme concentration, in
contrast to rmax.

MM eq.  plotted straight
line to describe the kinetics
of the enzyme under
study.

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Lineweaver-Burke plot
•MM eq.  converted to a linear
form
•plotting 1/r (y) vs 1/[S] (x)
•called a double reciprocal plot
or alineweaver-burke
•The equation of the line is:

•the slope is KM/Vmax

•the intercept on the 1/r axis is
1/rmax

Example

From series of batch runs
with a constant E
concentration, the
following initial rate data
were obtained as a
function of initial S
concentration:

Evaluate MM kinetic
parameters using
Lineweaver-Burk plot (do
not use deviated data
points from the MM model)

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Data:
S< 10mM  rmax=0.54

S increase up to l0 mM mM/min & Km=1.78 mM
the rate increased
All data  rmax=0.45

Further S increases (to mM/min & Km=1, 37 mM
15mM)  decreased the
initial reaction rate

may be due to substrate or
product inhibition

MM eq. does not
incorporate the inhibition
effects

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