k₁ k₂

E + S ES E + P
k₋₁
Fast Slow
 The Michaelis-Menten Approach
k1 k2
[E] + [S] [ES] [P] + [E]
k-1
Assumption: k-1 >> k2 i.e. the equilibrium of [E], [S] and [ES] is not
affected by k2:
k-1 [E] [S] KS = dissociation constant
KS = =
k1 [ES] [ES] = „Michaelis-Menten“ complex

Since we assume equilibrium it follows:

k-1 [ES]
[E] [S] k1 = [ES] k-1 solving for [E] = (1)
k1 [S]

In addition we know that: [E]total = [E] + [ES] (2)

This relationship is called the „enzyme conservation equation“

 k-1 [ES]
[E] = (1)
k1 [S]
[E]total = [E] + [ES] (2)
Solving equation (2) for [E] and substituting [E] in equation (1):

k-1
[E]total = [ES] (1 + ) (3)
k1 [S]
We also know that the velocity of the reaction equals:

v = k2 [ES] (4)

Solving equation (3) and (4) for [ES] and then substituting [ES] in
equation (3) with [ES] = v / k2 then yields:
 k2 [E]total k2 [E]total [S]
v= =
k-1 k-1
(1 + ) [S] +
k1 [S] k1

We define k-1/ k1 as KM, the Michaelis-Menten constant and the

maximal velocity as vmax = k2 [E]total

This simplifies the above equation to:

if [S] >> KM then v = vmax
vmax
if [S] = KM then v =
2

Therefore KM can be viewed as the substrate concentration with half-

maximal velocity (dimension M, typically mM to nM)
 Understanding Km and Vmax
What are Km and Vmax?
 Km - relates to affinity ; Vmax relates to efficiency
 Km tell how much substrate to use in an assay
 If more than one enzyme share the same substrate, KM
also will determine how to decide which pathway the
substrate will take
Vmax tells about pathways
 Rate limiting enzyme in pathway
 Km and Vmax can be used to determine effectiveness of
inhibitors and activators for enzyme studies and clinical
applications
 Catalytic Efficiency of Enzymes
For an enzyme that obeys the Michaelis-Menten kinetics:
vmax = k2 [E]total
k2 is also called kcat or turnover number because it reflects directly the
commitment to catalysis and therefore we can also write:
vmax
vmax = kcat [E]total or kcat =
[E]total
Michaelis-Menten Eq.:
When [S] << KM then [E] ~ [E]total

k2 [E]total [S]
v =
KM + [S]
kcat/KM is a 2nd order rate constant (M-1 sec-1)
kcat and as such reflects the efficiency of “E to
v = [E] [S] react with S”
KM
These consideration also allow us to determine how fast an enzyme
catalyzed reaction can proceed:
 Maximal velocity of an enzyme-catalyzed reaction
kcat k2 k1 k2
= =
KM KM k-1 + k2

In the case of efficient catalysis k2 >> k-1

This means that the Michaelis-Menten complex decays rapidly to
the product(s) and the back-reaction to free enzyme and substrate
are much slower (“enzyme is committed to catalysis”). In such a
case the equation above can be rewritten as:
Since k1 is the rate at which the Michaelis-Menten
complex forms, the limiting value for this rate
kcat constant is the rate of encounter of enzyme and
= k1
KM substrate, i.e. the rate limited by diffusion. This
rate is of the order 108-109 M-1 sec-1. Hence enzymes
that operate in this range have achieved maximal
velocity (catalase: 4 x 108 M-1 sec-1)
 Determination of Rates

• From steady-state measurements two enzyme parameters

are obtained:
1) The Michaelis-Menten Parameter (KM) which may be
equivalent to the enzyme-substrate dissociation
constant
2) kcat (turnover number) which may be a microscopic
rate constant or a combination of several

• To observe individual rates the approach to steady-state needs

to be observed (“ pre-steady-state kinetics”); the rates are
typically on the order of 1- 10-7 sec!
Lineweaver-Burk Plot
Hanes-Woolf Plot