Enzyme Kinetics: Michael-Menten Approach

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
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
graph will curve at higher

[S] & will level off at very
high [S]
The rate does not
depend on the [S] when
the [S] is high
Reaction rate changes
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.
the enzyme becomes

"saturated" and the
reaction rate reaches a
constant value doesn't
increase significantly as
yet more substrate is
added.
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
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.
Henry (1902) observed

this & proposed:
(2)
rmax & Km kinetic
parameters,
experimentally determined
Brown (1902) E forms a
complex with S. The
complex then breaks down
to the products,
regenerates free enzymes
(3)
(4)
Assumptions:
1. The total enzyme
concentration constant
during the reaction; [Eo] =
[ES]+[E]
2. The enzyme is very small
amount compared to the
amount of substrate the
formation of the complex
does not significantly deplete
the substrate.
3. [P] is so low that product
inhibition may be considered
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
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
From MM eq
Initial [S] increase reaction

goes faster; enzymes easier
to find S
All active sites occupied with
bound S or P saturated
[S]<<<[Km] double const,
double rate
rmax rate approached at
saturated [S]; rate does not
depend on [S]
Km: [S] required to produce
rate that is one-half of rmax
Km >> the weaker the
interaction between enzyme &
S
[S]=Km rate=one-half of
rmax
[S]<<Km rate depends
linearly on [S]; 2x[S] 2x
rate
[S]>>Km dependence of
rate on [S] approach a
max horizontal
line/independent; rmax
zero order kinetic
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.
Alternate Forms of the MM eq.
Where does KM & rmax come

in?
Estimate Km & rmax
asymptote
Km=f ([S])
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
if KM is high, the curve will

appear to spread out toward
the right, approaching Vmax
only at high substrate
concentration.
Km = rmax/2
rmax a property of the

enzyme, interaction of E &
S
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.
Km, depends on the

particular enzyme and
substrate being used 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.
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)
Data:
S increase up to l0 mM
the rate increased
Further S increases (to
15mM) decreased the
initial reaction rate
may be due to substrate or
product inhibition
MM eq. does not
incorporate the inhibition
effects
S< 10mM rmax=0.54

mM/min & Km=1.78 mM
All data rmax=0.45
mM/min & Km=1, 37 mM

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ENZYME KINETICS

Simplest enzymatic reaction 1st order

Real enzyme mechanism more complexes;

The reaction will start at a high

[S] very low a straight line

graph will curve at higher

the enzyme becomes

deals with the rate of enzyme reaction

Factors affecting the rate of

[S] low they are all bound to

Henry (1902) observed

If slower reaction (4) determines overall reaction rate of

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

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

Total enzyme contents:

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

Final rate eq:

Know as MM eq identical to (2)

Initial [S] increase reaction

Theory shows that KM is approximately equal to the

Alternate Forms of the MM eq.

Where does KM & rmax come

if KM is high, the curve will

rmax a property of the

Km, depends on the

the slope is KM/Vmax

From series of batch runs

S< 10mM rmax=0.54

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