Enzyme Kinetics - Inhibition

Types of Inhibition
• Competitive Inhibition
• Noncompetitive Inhibition
• Uncompetitive Inhibition
• Irreversible Inhibition
 Competitive Inhibition

Enzyme
S
I

In competitive inhibition,
the inhibitor competes
with the substrate for the
same binding site
 Competitive Inhibition
- Reaction Mechanism

E+S ES E+P
+
I
In competitive inhibition, the
EI inhibitor binds only to the
free enzyme, not to the ES
complex
General Michaelis-Menten Equation

Vmax,app [S]
v=
Km,app + [S]

This form of the Michaelis-Menten equation

can be used to understand how each type of
inhibitor affects the reaction rate curve
In competitive inhibition, only the apparent Km
is affected (Km,app> Km),

The Vmax remains unchanged by the presence of

the inhibitor.
.
Competitive inhibitors alter the
apparent Km, not the Vmax
- Inhibitor
Vmax
Reaction Rate

+ Inhibitor
Vmax
Vmax,app = Vmax
2
Km,app > Km

Km Km,app
[Substrate]
 The Lineweaver-Burk plot is
diagnostic for competitive inhibition
1 = Km,app 1
+ 1 Increasing [I]
v Vmax [S] Vmax

Km,app
1 Slope =
Vmax
v

1
Vmax

-1 1
Km,app
[S]
Relating the Michaelis-Menten equation, the v vs. [S]
plot, and the physical picture of competitive inhibition

Inhibitor
competes with S
substrate,
.

decreasing its I .

apparent affinity:
Km,app > Km Vmax
- Inhibitor

Reaction Rate
+ Inhibitor
E+S ES E+P Vmax
+ 2
I Km,app > Km
Formation
FormationofofEI EI Vmax,app = Vmax
complex
complex shifts
shiftsreaction
reaction
EI to
to the
theleft:
left:KKm,app > Kmm
m,app > K
Km Km,app
[Substrate]
 Example - Competitive Inhibition
NH2
Sulfanilamide is a competitive
inhibitor of p-aminobenzoic
folic acid acid. Sulfanilamides (also
known as sulfa drugs,
COOH discovered in the 1930s)
p-aminobenzoic acid were the first effective
NH2 systemic antibacterial
agents.
Because we do not make folic
acid, sulfanilamides do not
affect human cells.
SO2 NH2
sulfanilamide
Practical case: Methanol poisoning

A wealthy visitor is taken to

the emergency room, where
he is diagnosed with
methanol poisoning. You
are contacted by a 3rd year
medical student and asked
what to do? How would you
suggest treating this
patient?
Methanol (CH3OH) is metabolized to
formaldehyde and formic acid by alcohol
dehydrogenase. You advisethe third year
student to get the patient very drunk.
Since ethanol (CH3CH2OH) competes with
methanol for the same binding site on
alcohol dehydrogenase, it slows the
metabolism of methanol, allowing the
toxic metabolites to be disposed of before
they build up to dangerous levels. By the
way, the patient was very grateful and
decided to leave all their worldly
possessions to the hospital. Unfortunately,
after being released from the hospital, he
went to the casinos and lost everything he
had.
 .

Noncompetitive Inhibition
I I
S
Enzyme S Enzyme

the inhibitor
does not
S interfere with
I I
substrate
S binding (and
Enzyme Enzyme
vice versa)
 Noncompetitive Inhibition -
Reaction Mechanism
E+S ES E+P
+ + In noncompetitive
inhibition, the
I I inhibitor binds
enzyme
irregardless of
whether the
EI + S ESI substrate is bound
 Noncompetitive inhibitors decrease
.

the Vmax,app, but don’t affect the Km

Vmax - Inhibitor
Reaction Rate

Vmax,app
1
V
+ Inhibitor
2 max
1
V
2 max,app
Vmax,app < Vmax
Km,app = Km
Km [Substrate]
Km,app
 Why does Km,app = Km for
noncompetitive inhibition?
E+S ES E+P
+ + The inhibitor binds
equally well to free
I I enzyme and the ES
complex, so it doesn’t
alter apparent affinity

EI + S
of the enzyme for the
ESI substrate
The Lineweaver-Burk plot is diagnostic
for noncompetitive inhibition
1 = Km 1 1 Increasing [I]
+
v Vmax,app [S] Vmax,app

1 Slope =
Km
v Vmax,app

1
Vmax,app
-1 1
Km
[S]
Relating the Michaelis-Menten equation, the v vs. [S] plot,
and the physical picture of noncompetitive inhibition
I I
.

S
Enzyme S Enzyme
Inhibitor doesn’t interfere
with substrate binding,
Km,app = Km

S
I I
.

S Vmax - Inhibitor
Enzyme Enzyme

Reaction Rate
Vmax,app
E+S ES E+P 1 + Inhibitor
+ + Even at high
substrate levels, 1
V
V
2 max

Km,app >K
Vmax,app <m
Vmax
I FormationI inhibitor
of EI still binds,
2 max,app

Vmax,app
Km,app==V
Kmax
complex shifts
[E]t < reaction
[ES] m

to the left: VKmax,app < Vmax

m,app > Km
Km Km,app
EI + S ESI [Substrate]
Noncompetitive inhibitors
decrease the apparent Vmax, but
do not alter the Km of the
reaction
 Example of noncompetitive inhibition:
fructose 1,6-bisphosphatase inhibition by AMP
O O
-O -O
P O- P O-
fructose 1,6- O O
diphosphate H2 C CH2
fructose 6-
O
phosphate
H HO
H OH
OH H
Pi
fructose 1,6-
diphosphate
fructose 1,6- fructose 1,6- fructose 1,6-
bisphosphatase bisphosphatase bisphosphatase

E E.S E+P
AMP AMP AMP AMP

AMP AMP
O O
-O -O
P O- P O-
fructose 1,6- O O
diphosphate H2 C O CH2

H HO
H OH
OH H
fructose 1,6-
diphosphate
fructose 1,6- fructose 1,6-
bisphosphatase bisphosphatase

E.I E.S.I
Fructose 1,6-bisphosphatase is a key regulatory
enzyme in the gluconeogenesis pathway. High
amounts of AMP signal that ATP levels are low and
gluconeogenesis should be shut down while
glycolysis is turned on.
High AMP levels inhibit fructose 1,6-bisphosphatase
(shutting down gluconeogenesis) and activate
phosphofructokinase (turning on glycolysis).
Regulation of fructose 1,6-bisphosphatase and
phosphofructokinase by AMP prevents a futile cycle
in which glucose is simultaneously synthesized and
broken down.
 Uncompetitive Inhibition
Enzyme Enzyme
.

S
In uncompetitive
S
inhibition, the
Enzyme
I
inhibitor binds
I
only to the ES
complex
Enzyme

I S
 Uncompetitive Inhibition -
Reaction Mechanism
E+S ES E+P
+ In uncompetitive
I inhibition, the
inhibitor binds only
to the ES complex,
it does not bind to
ESI the free enzyme
 Uncompetitive inhibitors decrease
both the Vmax,app and the Km,app
.

Vmax,app < Vmax

Km,app < Km
Vmax - Inhibitor
Notice that at low substrate
Reaction Rate

concentrations,
uncompetitive inhibitors
Vmax,app have little effect on the
1
V + Inhibitor reaction rate because the
2 max
lower Km,app of the enzyme
1
V
2 max,app offsets the decreased Vmax,app

Km,app Km [Substrate]
Uncompetitive inhibitors decrease both the
Vmax,app and the Km,app of the enzyme

E+S ES E+P
+ Notice that
uncompetitive inhibitors
I don’t bind to the free
enzyme, so there is no
EI complex in the
reaction mechanism

ESI
 The Lineweaver-Burk plot is
diagnostic for uncompetitive inhibition
1 = Km,app 1 1
+
v Vmax,app [S] Vmax,app 1 Increasing [I]

=
Km 1
+
1 v
Vmax [S] Vmax,app
Km
Slope =
Vmax

1
Vmax,app

-1 1
Km,app
[S]
 Relating the Michaelis-Menten equation, the v vs. [S]
plot, and the physical picture of uncompetitive inhibition
Enzyme.

Enzyme

Vmax - Inhibitor

Reaction Rate
S
S
Enzyme
Vmax,app
I I 1
V
2 max
+ Inhibitor
Inhibitor 1
V Vmax,app < Vmax
increases Enzyme
2 max,app

the amount of Km,app< Km

enzyme bound
to substrate I S Km,app Km
.

[Substrate]
Km,app < Km

E+S ES E+P
+ Even at high
Formation
I of EI levels,
substrate
inhibitor binds,
complex shifts reaction
[E]t < [ES]
to the left: KVm,app >< V
Km
ESI
max,app max
Uncompetitive inhibitors
decrease the apparent Km of the
enzyme and decrease the Vmax of
the reaction
Example of uncompetitive inhibition: alkaline
phosphatase inhibition by phenylalanine
.

Alkaline Alkaline Alkaline

phosphatase phosphatase phosphatase

O O
O P O- -
O P O-
O O-
O
O-
P
O
O

-
-

Phe Phe

Alakaline
Phosphatase

O
O P O-
Phe O-
At alkaline pH, alkaline phosphatase catalyzes
the release of inorganic phosphate from
phosphate esters. It is found in a number of
tissues, including liver, bile ducts, intestine,
bone, kidney, placenta, and leukocytes.
Alkaline phosphatase plays a role in the
deposition of hydroxyapetite in osteoid cells
during bone formation. The function of
alkaline phosphatase in other tissues is not
known. Serum alkaline phosphatase levels are
important diagnostic markers for bone and
liver disease.
 Irreversible Inhibition
In irreversible
Enzyme inhibition, the
inhibitor binds to the
S enzyme irreversibly
O I through formation of
a covalent bond with
the enzyme ,
permanently
inactivating the
enzyme
Irreversible Inhibition - Reaction
Mechanism
E+S ES E+P
+ In irreversible inhibition,
I the inhibitor permanently
inactivates the enzyme.
The net effect is to remove
enzyme from the reaction.
EI Vmax decreases
No effect on Km
The Michaelis-Menten plot for an irreversible
.

inhibitor looks like noncompetitive inhibition

Vmax - Inhibitor
Reaction Rate

Vmax,app
1
V
+ Inhibitor
2 max
1
V
2 max,app Vmax,app < Vmax
Km,app = Km
Km [Substrate]
Km,app
 Irreversible inhibition is distinguished from
noncompetitive inhibition by plotting V max vs [E]t
.

tor
ibi
r Enzyme is
ito

Inh
t or
i inactivated
hib

ib
nh

ble
I until all of the
- In
Vmax

b le v e
i titi

rs i
r s
e pe irreversible
v

ve
R e om
+ nc inhibitor is
rr e
No
+I used up
[E]t

[E]t < [I] [E]t > [I]

[E]t = [I]
Irreversible inhibitors decrease
Vmax,app, but leave the apparent Km
unchanged. Irreversible
inhibitors differ from other types
of inhibitors because they
covalently modify the enzyme.
This results in the permanent
inhibition of the enzyme activity.
 Examples of Irreversible Inhibitors
• diisopropylphosphofluoridate
– prototype for the nerve gas sarin
– permanently inactivates serine proteases by
forming a covalent bond with the active site
serine
 Penicillin is a suicide inhibitor
R
O Penicillin
C
S CH3
H
H N

HC CH3

N COO-
C
H
O Strained
peptide bond R
O
glycopeptide C
glycopeptide H S CH3
transpeptidase transpeptidase
H N

HC CH3

N COO-
Ser OH Ser O C
H
H
O

Glycopeptide transpeptidase catalyzes the formation of cross-links between

D-amino acids in the cell walls of bacteria. This enzyme also catalyzes the
reverse reaction, the hydrolysis of peptide bonds. During the course of
hydrolyzing the strained peptide bond in penicillin, the enzyme activates the
inhibitor (penicillin), which then covalently modifies an active site serine in
the enzyme. In effect, the enzyme “commits suicide” by hydrolyzing the
strained peptide bond in penicillin.
Suicide inhibitors work by
“tricking” the enzyme into
activating the inhibitor, which
then forms a covalent bond with
the enzyme, leading to its
permanent inactivation.
 Summary-Enzyme Inhibition
• Competitive Inhibitor
– Binds to substrate binding site
– Competes with substrate
– The affinity of the substrate appears to be decreased
when inhibitor is present (Km,app >Km)
• Noncompetitive inhibitor
– Binds to allosteric site
– Does not compete with the substrate for binding to
the enzyme
– The maximum velocity appears to be decreased in
the presence of the inhibitor (Vmax,app <Vmax)
• Uncompetitive Inhibitor
– Binds to the enzyme only after the substrate has
bound
– The affinity of the substrate appears to be increased
and the maximum velocity appears to be decreased
when inhibitor is present (Km,app <Km, Vmax,app
<Vmax),
• Irreversible Inhibitor
– Covalently modifies and permanently inactivates the
enzyme