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Rates of Chemical Reactions Rates of Chemical Reactions

Enzyme kinetics is the study of rates of Enzyme kinetics is the study of rates of
reactions catalyzed by enzymes.
d[S] d[P]
v = S P
k
v
d[S]
dt

d[P]
dt dt dt
The reaction rate (velocity, v) can be
described in several ways.
Disappearance of substrate, S
Appearance of product, P
These equations relate velocity to These equations relate velocity to
concentration of reactants and products.
Consider the reaction catalyzed by
triose phosphate isomerase.
As [GAP] decreases, the [DHAP]
increases increases.
GAP DHAP
Reaction velocity can be thought of
as concentration vs. time.
Many enzymes react with substrates
in a nonlinear fashion.
The shape here
is hyperbolic. is hyperbolic.
Sh i di t Shape indicates,
in part, that E and
S combine to
form an ES
complex
S S E + S ES
KEY CONCEPTS KEY CONCEPTS
Simple chemical reactions are described Simple chemical reactions are described
in terms of rate constants.
The Michaelis-Menten equation describes The Michaelis-Menten equation describes
enzyme-catalyzed reactions in terms of
K d V K
M
and V
max
.
Rate equations describe chemical
processes.
A unimolecular reaction has a velocity
(rate) that is dependent on the ( ) p
concentration of only one substrate.
v = A P
k
k [A] here k has nits of sec
1
v A P
v = k [A], where k has units of sec
-1
Rate equations describe chemical
processes.
A bimolecular (second order) reaction
has a velocity (rate) that is dependent on y ( ) p
two substrate concentrations.
v = A + B P
k
k [A] [B] (or k [A]
2
or k [B]
2
) v = k [A] [B] (or k [A]
2
or k [B]
2
)
where k has units of M
-1
sec
-1
Many enzymes obey Michaelis-Menten Many enzymes obey Michaelis Menten
kinetics.
E + S ES E + P
k
1
k
2
E + S ES E + P
k
-1
Rate limiting step Rate limiting step
d[P]
P bl
v
d[P]
dt
k2[ES]
Problem:
[ES] is difficult to measure!
dt
What can we do?
Try to re express the rate Try to re-express the rate.
k
k
E + S ES E + P
k
1
k
-1
k
2
-1
O i t th t h l
d[ES]
One point that helps:
k
1
[E] [S] - k
-1
[ES] - k
2
[ES]
d[ES]
dt

Depletion of ES
Formation
of ES
Assume steady state equilibrium Assume steady state equilibrium.
For most of the duration of the reaction, ,
[ES] remains steady as substrate is
converted to product converted to product.
d[ES] d[ES]
dt
0
Michaelis Menten Equation Michaelis-Menten Equation
k
k
E + S ES E + P
k
1
k
-1
k
2
d[P]
-1
v
d[P]
dt
k2[ES]
= k
1
[E] [S] - k
-1
[ES] - k
2
[ES]
d[ES]
dt
0
dt
Derivation of the
Michaelis-Menten Equation
= k
1
[E] [S] - k
-1
[ES] - k
2
[ES]
d[ES]
dt
0
dt
Thus: k [E] [S] = [ES] (k + k ) Thus: k
1
[E] [S] = [ES] (k
-1
+ k
2
)
[E]
total
= [ES] + [E] Since
[E] = [E]
total
- [ES]
Substitute
here
Derivation of the
Michaelis-Menten Equation
k
1
([E]
total
[S] - [ES] [S]) = [ES] (k
-1
+ k
2
)
Divide both sides by k
1
K
M
[E]
total
[S] - [ES] [S] = [ES] (k
-1
+ k
2
)
k
1
[E]
total
[S] - [ES] [S] = [ES] K
M
Derivation of the
Michaelis-Menten Equation
[E]
total
[S] - [ES] [S] = [ES] K
M
Rearrange:
[E]
total
[S] = [ES] (K
M
+ [S])
[ES] = [E] [S] [ES] = [E]
total
[S]
K
M
+ [S]
M
[S]
Derivation of the
Michaelis-Menten Equation
v
d[P]
k2[ES]
k [E] [S]
V
max
v
dt
k2[ES]
k
2
[E]
total
[S]
K
M
+ [S]
v =
M
[ ]
V
max
[S]
K [S]
v =
The Michaelis-
Menten Equation
K
M
+ [S]
Menten Equation
The Michaelis-Menten equation is
hyperbolic.
V is where the reaction velocity reaches its plateau V
max
is where the reaction velocity reaches its plateau
K
M
is the substrate
concentration at V concentration at V
max
KEY CONCEPTS: Section 7 2 KEY CONCEPTS: Section 7-2
The kinetic parameters K k and The kinetic parameters K
M
, k
cat
, and
k
cat
/K
M
are experimentally determined.
K
M
and V
max
values can be derived for
M max
enzymes that do not follow the Michaelis-
Menten model Menten model.
The catalytic rate constant determines y
how quickly an enzyme can act.
k =catalytic rate k
cat
= catalytic rate
constant, turnover
k
t
=k
2
k
cat
k
2
k
cat
= V
max
/[E]
total
k
t
/K
M
indicates catalytic efficiency k
cat
/K
M
indicates catalytic efficiency.
Wh t li it th t l ti f ? What limits the catalytic power of an enzyme?
Electronic rearrangements during formation of the
transition state
Frequency of productive enzyme collision with
substrate with the maximumbeing the diffusion- substrate, with the maximum being the diffusion
controlled limit
Enzymes reach catalytic perfection when their
t i diff i t ll d rate is diffusion-controlled.
The Lineweaver-Burk plot linearizes
Michaelis-Menten kinetics data.
V
max
[S]
v =
K
M
+ [S]
Take the reciprocal of both sides:
v
1
=
K
M
V
+
[S]
1
V
1
v
V
max
[S]
V
max
The Lineweaver-Burk plot linearizes
Michaelis-Menten kinetics data.
Notice how the data are weighted heavily
here due to the linearization!
Not all enzymes fit the Michaelis-
Menten model.
Some enzymes have multiple substrates.
Not all enzymes fit the Michaelis-
Menten model.
Some enzyme-catalyzed reactions have
many steps. y p
Not all enzymes fit the Michaelis-
Menten model.
With allosteric enzymes, binding of a substrate
at one active site can affect the catalytic activity y y
of other active sites.
Not all enzymes fit the Michaelis-
Menten model.
Allosteric enzymes exhibit cooperativity.
Velocity plot is sigmoidal!
KEY CONCEPTS: Section 7 3 KEY CONCEPTS: Section 7-3
Substances that bind irreversibly to an Substances that bind irreversibly to an
enzyme can inhibit its activity.
A competitive inhibitor appears to
increase K
M
without affecting V
max
.
M
g
max
Transition state analogs can act as
competitive inhibitors competitive inhibitors.
Noncompetitive, mixed, and p
uncompetitive inhibitors decrease k
cat
.
Allosteric regulators can inhibit or activate Allosteric regulators can inhibit or activate
enzymes.
Some inhibitors act irreversibly. y
A suicide inhibitor
Competitive inhibitors bind to the
same site as the substrate.
Competitive inhibitors increase K
M
,
but do not affect V
max.
Transition state analogs often make
b tt i hibit th b t t l better inhibitors than substrate analogs.
Transition State
Transition State Analog
Noncompetitive inhibitors appear to
decrease V
max
and K
M.
Allosteric regulation can inhibit or
enhance enzyme activity.
Consider the example:
Phosphoenolpyruvate inhibits PFK by p py y
an allosteric feedback mechanism.