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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]
+ 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 - Inhibitor
Vmax
Reaction Rate
+ Inhibitor
E+S ES E+P Vmax
+ 2
I Km,app > Km
Formation
Formationof ofEI EI Vmax,app = Vmax
complex
complexshifts
shiftsreaction
reaction
EI totothe
theleft:
left:KKm,apm,appp >>KKmm 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
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
.
Vmax,app
1
V
+ Inhibitor
2 max
1 Vmax,app < Vmax
V
2 max,app
Km,app = Km
Km [Substrate]
Km,app
Why does Km,app = Km for
noncompetitive inhibition?
E+S ES E+P
+ + The inhibitor binds
I I
equally well to free
enzyme and the ES
complex, so it doesn’t
alter apparent affinity
EI + S ESI
of the enzyme for the
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,apVpmax,app> K<m
Vmax
I FormationI inhibitor
of EI still binds,
2 max,app
ESI m
[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 P O- 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
.
concentrations, uncompetitive
inhibitors have little effect on
Vmax,app the reaction rate because the
1
V + Inhibitor lower Km,app of the enzyme
2 max
1
offsets the decreased Vmax,app
V
2 max,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 don’t bind to
I 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
[Substrate]
Km,app < Km
E+S ES E+P
+ Even at high
I of
Formation EI levels,
substrate
inhibitor binds,
complex shifts reaction
[E]t < [ES]
to the left: KVm,app >< VKm
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
.
O O
O PO - -
OP O -
O
O-
O
O-
P
O
O
-
-
Phe Phe
Alakaline
Phosphatase
O
O P
Phe
-
O
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
S inhibitor binds to the
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, the
I 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
.
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
.
tor
ibi
r Enzyme is
ito
nh
tor
bi
hi b
inactivated
eI
hi
In
- In
Vmax
bl
i b le ive until all of the
rsi
rs etit
ve mp irreversible
ve
e
+ R nco
rre
inhibitor is
No
+I used up
[E]t
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