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Maud Menten
Leonor Michaelis
Enzyme Kinetics
What is it?
Enzymes are molecular machines as with any machine or mechanistic
process in nature, it is worthwhile to understand:
the individual steps taken by the machine in its process
the rate at which the machine can operate
the environment in which the machine best operates
k1
k2
k-1
Enzyme
(E)
Substrate
(S)
k-2
Enzyme-Substrate
Complex
(ES)
Enzyme
(E)
Product
(P)
reaction rate, v
ES complex
E+S
fast
slow
k1
k2
k-1
ES
k-2
E+P
reaction rate, v
Vmax
y=
ax
b+x
v=
Vmax[S]
Km + [S]
KM
E+S
fast
slow
k1
k2
k-1
ES
k-2
E+P
reaction rates are always measured
as initial velocities, before there is
appreciable depletion of substrate
or formation of product
approximation
E+S
fast
slow
k1
k2
k-1
ES
E+P
Equation 1
E+S
fast
slow
k1
k2
k-1
ES
E+P
breakdown
collect terms on right side equation
[ES] =
k1[E][S]
(k-1 + k2)
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E+S
[ES] =
fast
slow
k1
k2
k-1
ES
k1[E][S]
(k-1 + k2)
k1
(k-1 + k2)
1
KM
E+P
or
KM =
(k-1 + k2)
k1
[E][S]
[ES] =
KM
Equation 2
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E+S
fast
slow
k1
k2
k-1
ES
[E][S]
E+P
[ES] =
KM
Equation 2
[ES] =
([ET] [ES])[S]
KM
next page
E+S
[ES] =
[ES] =
fast
slow
k1
k2
k-1
ES
([ET] [ES])[S]
KM
[ET][S] [ES][S]
KM
[ES] =
E+P
collect terms
[ET][S]
KM + [S]
next page
E+S
[ES] =
fast
slow
k1
k2
k-1
ES
E+P
[ET][S]
remember Equation 1:
then
KM + [S]
v = k2[ES]
v
v
k2
[ES] =
[ET][S]
k2
solve for v
KM + [S]
k2[ET][S]
KM + [S]
Now, the maximum rate (Vmax) occurs when all enzyme bound to substrate.
That is, when [ET] = [ES]
Under this condition, Equation 1 becomes: Vmax = k2[ET]
substitute in Vmax
v=
Vmax[S]
KM + [S]
reaction rate, v
Vmax
y=
ax
b+x
v=
KM
Vmax[S]
Km + [S]
Vmax
E+S
fast
slow
k1
k2
k-1
ES
k-2
E+P
reaction rates are always measured
as initial velocities, before there is
appreciable depletion of substrate
or formation of product
approximation
E+S
fast
slow
k1
k2
k-1
ES
E+P
In general the rate of a reaction is dependent on the rate constant k2 and the
substrate concentration. Therefore v = k2[ES]
but, when substrate concentration is so high that all of the enzyme is saturated
then the enzyme is operating as fast as it can and Vmax = k2[ET] where ET is
the total Enzyme in the reaction ([ES]=[ET] when the enzyme is saturated).
Vmax is the maximal velocity of the enzyme
KM
KM is the substrate concentration at which the enzyme is rate is half its maximal
velocity (Vmax) or put another way:
KM is the substrate concentration where the enzyme is half-saturated.
reaction rate, v
Vmax
Vmax
KM
Vmax[S]
v=
Km + [S]
KM
k-2
k-1
Vmax[S]
v=
reaction rates are always measured
KM + [S]
as
initial
velocities,
before
there
is
approximation
depletion
of substrate
Km is actually a combined rate constant appreciable
that describes
the formation
and breakdown
or formation of product
of the ES complex
fast
E+S
k1 k 1
slow
k2
ES
(k
k-1-1 + k2)
1
KE
M
+ Por
KM =
(k-1 + k2)
k1
k1
(k-1 + k2)
k-1
k1
Note that when k1 > k-1, the formation of ES is favored and KM is small
when k-1 > k1, the dissociation if ES is favored and KM is large
that is, small KM indicates high affinity of enzyme for substrate
high KM indicates low affinity of enzyme for substrate
1
KM
Kcat
E+S
fast
slow
k1
k2
k-1
ES
E+P
Vmax
[ET]
= kcat
kcat
KM
Substrate
Kcat
(s-1)
KM
(M)
kcat/KM
(M-1s-1)
Catalase
H2O2
4 x 107
1 x 100
4 x 107
-lactamase
penicillin
2 x 103
2 x 10-5
1 x108
Hypothetical case:
You work for an industrial company that uses the enzyme Cellulase in the
formulation of laundry detergents.
It is your job to determine the kinetic attributes of 5 different Cellulase
enzymes to assess their suitability for use as an additive in laundry detergent.
Background
Cellulase is an enzyme that breaks down Cellulose into glucose molecules
Energy
O2
CO2 + H2O
glucose
cellulose
light
new shirt
surface
Your job
Carry out kinetic analysis of 5 cellulase enzymes
Cellulase 1
Cellulase 2
Cellulase 3
Cellulase 4
Cellulase 5
y=mx+b
[glucose]
m=d[glucose]/dt
time
[S]
[glucose]
[S]=4
[S]=3
[S]=2
[S]=1
time
1
2
3
4
5
6
v
0.001
0.002
0.004
0.007
0.010
0.013
v=
rate, v
o
o
o
o
Vmax
o
o
o
Vmax
o
o
o
o
o
[substrate], [S]
KM
Vmax[S]
KM + [S]
So,
Having generated a Michaelis-Menten Plot specific for the Cellulase #1:
You now know the Vmax for the enzyme.
You can now determine KM for the enzyme since KM is the substrate concentration
at 1/2Vmax
Also, you can calculate Kcat since you know how much enzyme you used in the assay
and
Vmax
Kcat =
[ET]
Enzyme
kcat
(s-1)
KM
(M)
kcat/KM
(M-1s-1)
Cellulase 1
4 x 104
1 x 10-2
4 x 106
Cellulase 2
1 x 102
4 x 10-4
3 x 105
Cellulase 3
2 x 105
4 x 10-2
5 x 106
Cellulase 4
4 x 101
4 x 10-3
1 x 104
Cellulase 5
2 x 103
2 x 10-5
1 x108
Therefore, you would report that Cellulase #5 is the most active enzyme
Note that Cellulase #1 has a higher turnover number (kcat) than Cellulase #5
But Cellulase #5 has a much higher affinity for substrate (KM).
Overall, based on the specificity constant (kcat/KM) Cellulase #5 is the most active
enzyme.
E+S
fast
slow
k1
k2
k-1
ES
E+P
Recommended Reading
Stryer 8th Edition
Ch 8 Enzymes: Basic Concepts and Kinetics Pgs 216-245.
Stryer 7th Edition
Ch 8 Enzymes: Basic Concepts and Kinetics Pgs 219-248.
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