Biochemistry I
Lecture 18
February 25, 2013
Lecture 18: Enzyme Kinetics & Intro to Inhibitors.
Why determine KM and kCAT?
A. Mechanistic Information: Provide mechanistic information about particular enzyme-substrate pairs.
1. The active site region of Trypsin is shown below.
i) What is the role of His57 and Asp189 in the catalytic mechanism?
ii) Sketch the kCAT as a function of pH.
iii) Sketch the KM as a function of pH.
Asp102
O
Asp189
O
His57
pKa=7.0
O
HN
+
NH3
pKa=5.0
O
N
O H
HN
Ser195
H
N
O
2. The three substrates shown on the right were presented to
trypsin. Their structures and measured KM and kCAT values
are given.
i) Explain the differences in KM of A versus B:
Substrate
kCAT
10 M
1000 s-1
100 M
1000 s-1
100 M
100 s-1
+
A
NH3
H3N
H
N
+
B
O
O
CH3
O
+
NH3
ii) Which type of bond is a serine protease more efficient
at cleaving, an amide or an ester? (Note- ester
hydrolysis of the acyl-enzyme always occurs in phase
II of the reaction.)
KM
H3N
H
N
+
O
CH3
O
C
O
+
NH3
O
H3N
O
+
O
O
CH3
[S]
1
0.3M
B. Predictive Information: An enzyme has a kcat v  k E
 105
 1 nmole
i
cat t
5
of 10 /sec and a KM of 0.1 μM towards a certain
K M  [S]
sec
0.1M  0.3M
substrate. One (1) nmole of enzyme is mixed
1
0.3M
nmole
 105
 1 nmole
 1  105
( 0.75)
with 0.3 μM substrate in a volume of one ml,
sec
0.4 M
sec
what is the initial rate of the reaction (dP/dt)?
moles
moles
M
 10 4
 0.75  7.5  10 5
 7.5  10 2
sec
sec
sec
1
Biochemistry I
Lecture 18
February 25, 2013
Enzyme Inhibitors
Studies on Inhibitors are useful for:
1. Mechanistic studies to learn about how enzymes interact with their substrates.
2. Understanding the role of inhibitors in enzyme regulation.
3. Drugs if they inhibit aberrant biochemical reactions:
 penicillin, ampicillin, etc.:interfere with the synthesis of bacterial cell walls
4. Understanding the role of biological toxins.
 Amino acid analogs - useful herbicides (i.e.
acetylcholine
roundup)
esterase
OH
 Insecticides - chemicals targeted for insect
+
O
CH3 (Serine esterase)
+
H3N
H3N
choline
nervous system.
Acetylcholine
HO
O
Molecular Mechanism of Inhibitors:
A: Suicide Inhibitors
 Inhibitor binds and forms a covalent bond with the
enzyme, inactivating it.
 Nerve gas sarin modifies the active site serine in the
serine esterase acetylcholine esterase.
B: Competitive Inhibition
1. Inhibitor binds to the same site on
HO
O
the enzyme as the substrate.
H
H
2. Inhibitor ONLY binds to the free
H
H
enzyme.
3. Inhibitor usually is structurally
HO
O
very similar to the substrate. For
Succinate
example, succinate is the normal
HO
O
substrate
for
the
enzyme
H
H
succinate
dehydrogenase.
Malonate
is
an
effective
HO
O
competitive inhibitor of this
enzyme.
Malonate
4. The inhibitor can’t undergo the chemical reaction.
C: Mixed Inhibition:
In this case the inhibitor binds to both
[E] and [ES]. The binding site of the
inhibitor is not at the active site.
However, the inhibitor binding causes
a change in the conformation of the
protein that affects either substrate
binding, the chemical step or both.
 Allosteric Activators increase
substrate binding and/or the rate of
the chemical step (kcat).
 Allosteric
inhibitors
reduce
substrate binding and/or the rate of
the chemical step.
Both VMAX and KM can be altered by
mixed inhibitors since the precise
geometry of the active site is altered
when the inhibitor is bound.
2
H3C
O
H3C
P O
CH3
sarin F O
(nerve gas)
O
H
H
HO
O
H3C
P O
CH3
O
Serine
HO
H3C
O
Serine
CH3
acetic
O acid
Biochemistry I
Lecture 18
February 25, 2013
D. Measuring Inhibitor-Enzyme Affinity:
This can be done by measuring the effect of the presence of the
inhibitor on the enzyme rate constants, KM and VMAX. Two sets
of experiments are performed:
1. Substrate is varied in the absence of the inhibitor, as you
would normally do to obtain KM and VMAX.
2. Substrate is varied in the presence of a known, and
constant, amount of inhibitor.
Competitive Inhibitor: A competitive inhibitor reduces the
amount of [E] by the formation of [EI] complex. The inhibitor
cannot affect the [ES] complex after it has formed since the
inhibitor can no longer bind.
E
+
(ES)
S
EP
(EI)
There are two anticipated consequences of this binding mode on
the steady-state kinetics:
1. VMAX is unchanged: At high levels of substrate all of the
inhibitor can be displaced by substrate, and ET=[ES].
2. The apparent KM is increased: It requires more substrate to
reach 1/2 maximal velocity because some of the enzyme is
complexed with inhibitor.
A
B
D
C
E
ES
EI
[S] < KM
A'
[S] = KM
B'
[S] > KM
C'
[S] >> KM
D'
6
5
4
vi
3
2
1
C
B
C'
A
B'
A'
[S]
3
w/o Inh
D
+ Inh
D'