Enzymes
OC
Hi, Everybody!
Intended learning
outcomes(ILO)
1. Compare reversible competitive to non competitive
enzyme inhibitors and others acting as irreversible enzyme
inhibitors.
2. List some examples of drugs acting by competitive enzyme
inhibition and others acting as irreversible enzyme
inhibitors.
Objectives
Enzymes as
Biological
Catalysts
Enzyme
Kinetics
The
Properties of
Enzymes
Enzyme
classification
Enzyme
Inhibition
Regulation of
enzyme
activity.
Applications
of Enzyme
Action
Enzymes Inhibitors
(reduce the reaction rate via a non substrate
molecule)
Enzymes inhibitors
Enzyme inhibitors
Reversible
Competitive
Non
competitive
Irreversible
Non
competitive
Reversible inhibitors
• They bind to enzymes non covalent bonds.
• Characterized by rapid dissociation of the
enzyme inhibitor complex.
Enzyme inhibitors
Competitive
Non
competitive
• Compete with
the substrate
for the active
site.
• Bind to the
enzyme at
sites other tan
the active site.
Competitive inhibitors
The Inhibitor is structurally similar to the substrate .
Competes with the substrate for the active site of the enzyme.
The inhibitor binds to E & forms an [EI] complex at the active site.
No product is formed
Competitive
Competitive inhibitors
Inhibition can be
overcome if [S] is very
high i.e. [S] is more than
[I]
No effect on Vm as excess substrate will
displace the inhibitor and the enzyme will
work at its
maximum rate
Competitive inhibitors
Increased Km because it takes more
substrate to half
saturate the enzyme.
For Lineweaver-Burk plots, Y intercept is the
same regardless of whether the inhibitor is
present or absent, but the slope differ between
the two lines(different Km)
Examples
Classical example
is malonic acid
inhibition of
(succinate
dehydrogenase
enzyme) of Kreb’s
cycle.
Examples and clinical use
DRUG
SIMILAR
TO(inhibit)
Uses
sulfonamides
Para-amino
benzoic acid
Antibacterial
drug
Dicumarol
Vitamin K
Anticoagulant
Examples and clinical use
DRUG
SIMILAR
TO(inhibit)
Uses
Methotrexate
Folic acid
(-Dihydrofolate
reductase(
Anticancer
Ethanol
Methanol
Alcohol
dehydrogenase
Methanol
toxicity
Statin
Inhibit HMGCOA reductase
Inhibit
cholesterol
Folic acid cannot be synthesized which is
essential for bacterial growth.
Methorexate (Anticancer drug)
Structural
analogue of
folic acid
Inhibit
dihydrofolate
reductase
Inhibit DNA
,RNA synthesis
Methorexate (Anticancer drug)
Cancer cells which are rapidly dividing are very
sensitive to treatment by methotrexate(antitumor
drug)
One problem of this drug is that affect other rapidly
dividing cells as bone marrow and mucosal cells
causing severe anemia and mucosal ulceration.
Ethanol
Methanol
Converted into
acetaldehyde which is
less toxic
Converted into
formaldehyde which is
toxic
Ethanol injected
intravenously to treat
methanol toxicity to
prevent damaging
effects of formaldehyde
Causes tissue damage
and blindness
Competitive inhibitors
NON-COMPETITIVE INHIBITION...
Inhibitor binds to the E, forms an
[EI] complex not at the active
site(Not affect the affinity of
the enzyme to substrate) .
Inhibitor often have no structural
similarity to substrate
Inhibition NOT reversed by
increasing [S].
Inhibitor can bind the enzyme before or
after substrate binding
E+S↔ 𝑬𝑺 + 𝑰 → 𝑬𝑺𝑰(𝒊𝒏𝒂𝒄𝒕𝒊𝒗𝒆)
E+I↔ 𝑬𝑰 + 𝑺 → 𝑬𝑺𝑰(𝒊𝒏𝒂𝒄𝒕𝒊𝒗𝒆)
Km not changed ,Vm decreased
Reversible Non
competitive
inhibitors
Inhibition can
be reversed by
dialysis of the
inhibited
enzyme.
Reverse
non
competitive
inhibition is
rare.
For Lineweaver –
Burk plot, lines for
inhibited reaction
converge on the X
axis with those for
the uninhibited
reaction
IRREVERSIBLE INHIBITOR
(Enzyme poison)
Inhibitor combines
or destroy a
functional group on
the enzyme that is
essential for the
activity
The binding is so
tight usually
covalent that they
are not dissociated
E+I→ 𝑬𝑰
The kinetics of
irreversible
inhibitors are
similar to reversible
non competitive
inhibitors(Decrease
Vmax)
IRREVERSIBLE INHIBITOR
Km is Same & Vmax are Lowered
It resembles enzyme Kinetics of non
competitive inhibitors
No competition between substrate
and inhibitor because the inhibitor
has no structural resemblance to
the substrate.
Increase of substrate not relieve the
inhibition.
Examples of irreversible non
competitive inhibitors
1-Alkylating agents like
iodoacetamide (bind to SH’s).
2-heavy metals (silver &
mercury) bind to -SH’s.
3-cyanide inhibit
mitochondrial
cytochrome oxidase.
4-Fluoride inhibit
enolase enzyme
5- Penicillin is an
antibiotic , inhibits
bacterial transpeptidase.
6-Nerve gas and organophosphorus on
cholinesterase.
7-Aspirin as anti-platelet
aggregator on cyclooxygenase
Inhibit prostaglandins
and thromboxane
synthesis.
Antimetabolites
• Block one or more of the metabolic
pathway involved in DNA
synthesis
• Used in treatment of cancer.
Organophosphorus
Compounds(diisopropylfluorophosphate=
Poison
DIPF)
nerve gas
Form covalent bond with a serine residue
in the active site of choline esterase
Cause neurotoxicity
When choline esterase is inactivated,
acetyl choline, the neuromuscular
transmitter, persists and this leads to
muscle paralysis and death.
Suicide inhibitors
• A special class of irreversible inhibitor
Inhibitor is structural analogue
to the substrate on which the
enzyme act giving product
The products bind
irreversibley with the enzyme
The product inactivate the
enzyme
Hypoxanthine xathine oxidase
Xanthine
xathine oxidase
Uric acid
-
Allopurinol(suiccide subtrate):
used in treatment of GOUT that result
from increased uric acid level in blood
N
N
Allopurinol
as
suicide
enzyme
inhibitor
Allopurinol is xanthine oxidase inhibitor
Allopurinol is similar to hypoxanthine in structure
Allopurinol is a substrate for xanthine oxidase, but the product binds so
tightly to enzyme
Xanthine oxidase enzyme is now unable to act on normal substrate.
Uric acid production is diminished and xanthine and hypoxanthine levels in
the blood rise.
Enzyme Inhibition (Mechanism)
Equation and Description
Cartoon Guide
I
Competitive
I
Reversible Non-competitive
Substrate
E
S
S
E
I
Compete for
Inhibitor active site
E + S←→ ES → E + P
+
I
↓
↑
EI
[I] binds to free [E] only,
and competes with [S];
increasing [S] overcomes
Inhibition by [I].
S
I
I Irreversible Non-competitive
S
E
I
I
Different site
E + S←→ ES → E + P
+
+
I
I
↓
↑
↑↓
EI + S →EIS
S
I
E + S←→ ES → E + P
+
I
↓
↑
EIS
[I] binds to [E] tightly
[I] binds to free [E] or [ES]
only, destroy functional
complex; Increasing [S] can
not overcome [I] inhibition. group increasing [S] favors
the inhibition by [I].
Enzyme Inhibition (Plots)
I
Competitive
I
Reversible Non-competitive
Direct Plots
Vmax
vo
Double Reciprocal
Vmax
vo
I
Km Km’
I
[S], mM
Km = Km’
Vmax decreased
Km unchanged
1/vo
1/vo
1/Km
I
1/ Vmax
1/[S]
Intersect
at X axis
1/Km
Vmax’
[S], mM
Vmax unchanged
Km increased
Intersect
at Y axis
I Irreversible Non-competitive
I
1/ Vmax
1/[S]
Vmax decreased