Chapter 2 P-2 Enzyme-Inhibition 1
Chapter 2 P-2 Enzyme-Inhibition 1
• 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]
+ Inhibitor
Vmax
2
Vmax,app = Vmax
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
Km,app
1
[S]
Relating the Michaelis-Menten equation, the v vs. [S]
plot, and the physical picture of competitive inhibition
Inhibitor
competes with
substrate,
decreasing its .
apparent affinity:
Km,app > Km Vmax
- Inhibitor
Reaction Rate
+ Inhibitor
Vmax
2
Km,app > Km
Formation
FormationofofEI EI Vmax,app = Vmax
complex
complex shifts
shiftsreaction
reaction
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.
SO2NH2
sulfanilamide
Practical case: Methanol poisoning
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
1 Slope =
Km
v Vmax,app
1
Vmax,app
-1
Km
1
[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
1
V
+ Inhibitor
Even at high 2 max
substrate levels, 1
V Km,app > K< mVmax
Vmax,app
Formation inhibitor
of EI still binds, 2 max,app
Vmax,app
Km,app== V
Kmmax
complex shifts
[E]t < reaction
[ES]
Vmax,app < Vmax Km Km,app
to the left: Km,app > Km
[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
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
Notice that at low substrate
concentrations,
uncompetitive inhibitors
have little effect on the
reaction rate because the
lower Km,app of the enzyme
offsets the decreased Vmax,app
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
Km,app
1
[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
[Substrate]
Km,app < Km
Even at high
Formation of EI levels,
substrate
complex shiftsinhibitor binds,
reaction
[E]t < [ES]
to the left: KVm,app > Km
max,app < Vmax
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
.
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 Vmax vs [E]t
Enzyme is
inactivated
until all of the
irreversible
inhibitor is
used up
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