Sandipayan_enzyme inhibition_seminar

Presented by
Sandipayan Dutta
MSc Biotechnology
2nd Semester
Year-2014.
Seminar On
Enzyme Inhibitors.

What is an Enzyme?
Enzymes are specialized proteins that act as catalysts; they
speed up chemical reactions by lowering the activation
energy required.
Enzymes are NOT used up during a reaction.
Enzymes are specific to a particular substrate (reactant).
All enzymes are proteins with a very specific 3-D shape.
A substrate is the molecule that the enzyme acts upon. The
3-D shape of the enzyme fits a substrate molecule like a lock
and key – a perfect fit.
Are specific for what they will catalyze
Are Reusable
End in –ase
-Sucrase, Lactase, Maltase

How do enzymes Work?
Enzymes work by weakening bonds which lowers activation
energy.

Free
Energy
Progress of the reaction
Reactants
Products
Free energy of activation
Without Enzyme
With Enzyme

Induced-Fit Hypothesis
Enzymes are flexible molecules and research shows that enzymes will
often change the shape of the active site very slightly so that it is an
even more precise fit for the substrate.
This phenomenon is known as the induced-fit model of enzyme
activity.
A change in the configuration of an enzyme’s active site (H+ and ionic bonds
are involved).
Induced by the substrate.

Lock & Key model of enzyme
Lock and Key Analogy: lock = enzyme, key =
substrate.

No. Classification Biochemical Properties
1 Oxidoreductases
Act on many chemical groupings to add or remove hydrogen atoms.
oxidases,dehydrogenases,reductases
2 Transferases
Transfer functional groups between donor and acceptor molecules.
Kinases are specialized transferases that regulate metabolism by
transferring phosphate from ATP to other
molecules.AST,ALT,hexokinase
3 Hydrolases Add water across a bond, hydrolyzing it.pepsin,trypsin
4 Lyases
Add water, ammonia or carbon dioxide across double bonds, or
remove these elements to produce double
bonds.arginosuccinase,fumarase
5 Isomerases
Carry out many kinds of isomerization: L to D isomerizations, mutase
reactions (shifts of chemical groups) and
others.racemases,epimerase,cis-trans isomerase
6 Ligases
Catalyze reactions in which two chemical groups are joined (or
ligated) with the use of energy from ATP.
All synthetases (synthase)

What Affects Enzyme Activity?
Three factors :
1. Environmental Conditions
2. Cofactors and Coenzymes
3. Enzyme Inhibitors

1. Environmental Conditions
1. Extreme Temperature are the most
dangerous
- high temps may denature (unfold) the
enzyme.
2. pH (most like 6 - 8 pH near neutral)
3. Ionic concentration (salt ions)

2. Cofactors and Coenzymes
Inorganic substances (zinc, iron) and vitamins
(respectively) are sometimes need for proper
enzymatic activity.
Example:
Iron must be present in the quaternary
structure - hemoglobin in order for it to
pick up oxygen.

3.Enzyme Inhibitors
Inhibition –
means the prevention or reduction of function.
Enzyme inhibitors are molecules that interact in
some way with the enzyme to prevent it from working in
the normal manner.
Since blocking an enzyme's activity can kill a
pathogen or correct a metabolic imbalance, many drugs are
enzyme inhibitors. They are also used as herbicides and
pesticides. Not all molecules that bind to enzymes are
inhibitors; enzyme activators bind to enzymes and increase
their enzymatic activity.

Why End a Good Thing?
Inhibit means stop!
If enzyme action is critical to normal health, then
why would we want to stop it?
Energy Efficiency – It requires energy to build
molecules or break them down.
Build up of Toxins – Some products are great
until their concentration gets too high – then they
become toxic to the cell. (Too much of a good
thing!)

NO MATTER HOW LARGE THE SUBSTRATE CONCENTRATION,REACTION RATE CAN NEVER EXCEED
VMAX.
Km IS THE SUBSTRATE CONCENTRATION AT WHICH REACTION RATE IS HALF MAXIMAL.
Km REFLECTS THE BINDING AFFINITY OF THE ENZYME FOR THE SUBSTRATE; THE HIGHER THE
AFFINITY, THE SMALLER IS KM.
© Copyright. Sandipayan Dutta. 2014. All
rights reserved.

Vo = VMAX
[S]
[S] + KM
Vo = VMAX
[S]
[S] + KM
Km IS CALLED MICHAELIS CONSTANT & IT IS INDEPENDENT OF ENZYME CONC.
Km IS CONSTANT FOR ENZYME.
IT IS EXPRESSED IN moles/L.
MICHAELIS-MENTEN EQUATION
LINEWEAVER-BURK PLOT OR THE
DOUBLE RECIPROCAL PLOT.
USEFUL FOR EXPERIMENTAL DETERMINATION
OF KM AND VMAX
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Enzyme inhibitors types
• Competitive
• Uncompetitive
• Non-competitive
• Mixed
• Partial
• Substrate
• Allosteric
• form strong covalent
bonds with the
enzymes, rendering it
inactive. This effect
can’t be overcome by
increasing the
concentration of the
substrate.
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1. Reversible
E + I E.I
Usually from non-covalent interaction with enzyme.
2. Irreversible
E + I E.I
Usually from covalent bond with enzyme.
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Major classes of enzyme inhibitor

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Reversible Inhibitors
Can bind either to free enzyme or enzyme-
substrate complex (usually non-covalently).

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Classification of Reversible Inhibitors
• Competitive – bind only to free enzyme
• Uncompetitive – bind only to enzyme-substrate
Complex
• Noncompetitive – bind equally well to free enzyme and
enzyme-substrate complex
• Mixed – binds to both free enzyme and enzyme substrate
complex, but with different affinities.

Partial Inhibition : The inhibition is partial, where Linewever-Burk plot would be
linear.
Substrate Inhibition : Substrate inhibition occurs when a molecule of substrate
binds to one site on the enzyme and then another molecule of substrate binds to a separate
site on the enzyme to form a dead-end complex.
Allosteric inhibitor : binds to the enzyme at a site distinct from the substrate binding
site. It is usually reserved for the enzyme, influences conformational changes which may
alter the binding characteristics of the enzyme for the substrate or the subsequent reaction
characteristics(or both).

Feed back inhibition
Feedback inhibition occurs when there is a chain of reactions that
must occur in order to achieve a final product.
When enough product has been made, some of the surplus product
goes back to an enzyme earlier in the chain and inhibits it.
When the surplus product is used up by the cell – the product acting
as an inhibitor will be consumed and open the pathway again.

Competitive Inhibition
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• Km = [S] when V = (1/2) Vmax
• In the presence of a competitive inhibitor, more
substrate is required to reach the uninhibited
Velocity
• But, given high enough [S], the same Vmax
can still be reached
Why does a competitive inhibitor have
these effects ?
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• “When stress is applied to a system at chemical
equilibrium, the equilibrium will shift in the
direction that tends to relieve or counteract that
stress.”
• A competitive inhibitor reduces [E]
 Equilibrium shifts toward [E]
[E] [S]
= K’m increases
[ES]
Why does a competitive inhibitor
have these effects?
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The Lineweaver-Burk plot is
diagnostic for competitive inhibition
Slope =
Km,app
Vmax
1
Vmax
-1
Km,app
1
[S]
Increasing [I]
1
v
v
=1
Vmax
Km,app
Vmax
1+
[S]
1
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Examples of competitive inhibitors
2,3-biphosphoglycerate
Inhibits its own formation by inhibiting biphosphoglycerate mutase
 Metabolic regulation by product inhibition.
Sulphonamides: widely used in medicine to limit bacterial growth
Antifreeze: ethylene glycol competes for active site of alcohol
dehydrogenase
Ethylene glycol is metabolized to oxalic acid, which crystallizes in kidneys and
causes renal failure
Can be treated by alcohol infusion
A new treatment, approved by the FDA in December 1997, uses the alcohol
dehydrogenase inhibitor 4-methylpyrazole (trade name = Antizol). Unlike
ethanol, 4-methylpyrazole is not a substrate for the enzyme, is therefore not
metabolized.

Examples of competitive
inhibitors
• Malonate vs succinate
Enzyme: succinate
dehydrogenase
• Krebs and his colleagues
used malonate to
investigate the TCA cycle
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DDT Insecticide
• Insecticide that effects
ATPase activity
– ATPase makes ATP from ADP
• DDT is a competitive
inhibitor that binds with the
active site on ATPase, thus
stopping the synthesis of ATP
in the mitochondria in the
body
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Non Competitive Inhibitors
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• E and E·S are bound equally by the noncompetitive
Inhibitor
• Amount of uninhibited enzyme is decreased, so
V’max is decreased
• E vs. E·S equilibrium is not altered, so K’m is
Unchanged
• Amount of substrate required to reach ½ Vmax is
unchanged, so K’m is unchanged
Why does a noncompetitive inhibitor
have these effects?
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The Lineweaver-Burk plot is diagnostic
for noncompetitive inhibition
v
=1
Vmax,app
Km
Vmax,app
1
+
[S]
1
Slope =
Vmax,app
Km
1
Vmax,app
-1
Km
1
[S]
Increasing [I]
1
v
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Examples of noncompetitive inhibitors
• Heavy metals like lead, mercury (breaks disulfide bonds),
chromium will act as non-competitive inhibitors
• Mono-amine oxidase (MAO) inhibitors that are used as anti-
depressants: They covalently react with the enzyme in the liver
and effectively remove it.
– There are many potent drug interactions with MAO
inhibitors. One of these is tyramine, a compound that is
present in red wine and aged cheeses
– MAO inhibitors and tyramine (blocks neurotransmitter
reuptake in the brain)  hypertensive crisis
– Patients are still subject to hypertension for as long as two
weeks after discontinuing the drug
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Difference between competitive & non competitive inhibitors

Uncompetitive Inhibitors
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• Only E·S complex is bound by the uncompetitive
Inhibitor
• Decrease in E·S means less active enzyme, so
apparent maximal velocity (V’max) decreases as well
• E vs. E·S equilibrium shifts toward E·S
[E] [S]
= K’m decreases
[ES]
Why does an uncompetitive inhibitor
have these effects?
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The Lineweaver-Burk plot is diagnostic for
uncompetitive inhibition
v
=1
Vmax,app
Km,app
Vmax,app
1
+
[S]
1
=
Vmax
Km
Vmax,app
1
+
[S]
1
1
Vmax,app
-1
Km,app
1
[S]
Increasing [I]1
v
Slope =
Vmax
Km
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Examples of uncompetitive inhibitors
• Lithium and the phosphoinositide cycle: an example of
uncompetitive inhibition and its pharmacological consequences
Nahorski SR, et al Trends Pharmacol Sci. 1991 Aug;12(8):297-
303
– The ability of lithium to exert profound and selective
psychopharmacological effects to ameliorate manic-depressive
psychosis has been the focus of considerable research effort.
– There is increasing evidence that lithium exerts its therapeutic
action by interfering with polyphosphoinositide metabolism in
brain and prevention of inositol recycling by an uncompetitive
inhibition of inositol monophosphatase
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• Many successful pesticides and drugs are tight-binding inhibitors,
but these are difficult to design because of the need to deliver and
maintain concentrations at least 1000-fold higher than the inhibition
constants
• A few pesticides are uncompetitive inhibitors, the best-known
example being the herbicide N-phosphonomethylglycine, commonly
known as glyphosate or Roundup, an uncompetitive inhibitor of 3-
phosphoshikimate 1-carboxyvinyltransferase.
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Kamali and Rawlins, Biopharmaceutics & Drug Disposition, 13(6), 403-409
• The effects of probenecid on zidovudine (a potent
HIV inhibitor) glucuronidation were investigated, in
vitro, using human liver microsomal preparations
• The presence of probenecid:
– reduced the Vmax for zidovudine glucuronide
formation by more than 60 %
– reduced Km by 47 %
– uncompetitive inhibition
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Enzyme Inhibition (Mechanism)
I
I
S
S
S I
I
I II
S
Competitive Non-competitive Uncompetitive
Different site
Compete for
active siteInhibitor
Substrate
CartoonGuideEquationandDescription
[I] binds to free [E] only,
and competes with [S];
increasing [S] overcomes
Inhibition by [I].
[I] binds to free [E] or [ES]
complex; Increasing [S] can
not overcome [I] inhibition.
[I] binds to [ES] complex
only, increasing [S] favors
the inhibition by [I].
E + S→ES→E + P
+
I
↓
EI
←
↑
E + S→ES→E + P
+ +
I I
↓ ↓
EI+S→EIS
←
↑ ↑
E + S→ES→E + P
+
I
↓
EIS
←
↑
E
I
S
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Km
Enzyme Inhibition (Plots)
I II Competitive Non-competitive Uncompetitive
DirectPlotsDoubleReciprocal
Vmax Vmax
Km Km’ [S], mM
vo
[S], mM
vo
I I
Km [S], mM
Vmax
I
Km’
Vmax’Vmax’
Vmax unchanged
Km increased
Vmax decreased
Km unchanged Both Vmax & Km decreased
I
1/[S]1/Km
1/vo
1/Vmax
I
Two parallel
lines
I
Intersect
at X axis
1/vo
1/Vmax
1/[S]1/Km 1/[S]1/Km
1/Vmax
1/vo
Intersect
at Y axis
= Km’
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Partial inhibitors
• In some situations, the enzyme can still work with the inhibitor
bound, but at a reduced rate
Activity of the enzyme can not be driven to zero...
• To be sure if the compound is a partial inhibitor or not, one
should pay attention to experimental conditions
• Lineweaver-Burk plot would be linear, BUT secondary plots of
intercept or slope vs [I] will not be linear
• Dose-response curves (DRC) and Dixon plots are also different
for partial inhibitors
– At high [I]  there is a residual fractional activity in DRC
– Dixon plots are not linear but hyperbolic
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Mixed Inhibition
1. Always possible
2. Not possible with uncompetitive
inhibitors
3. Not possible with competitive
inhibitors
4. Not possible with competitive
or uncompetitive inhibitors
A noncompetitive inhibitor is capable of all four
reactions, but the classical noncompetitive inhibitor,
as opposed to a mixed one, is a special case. With
these inhibitors Ks and Ks' are equal to each other, as
are Ki and Ki' © Copyright. Sandipayan Dutta. 2014. All
rights reserved.

Mixed Inhibition
When [E]0<< [I]0:
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allosteric activator
substrate cannot bind
allosteric inhibitor
allosteric
binding
site vacant;
active site can
bind substrate
active site altered,
substrate can bind
allosteric binding site vacant
enzyme active site
active site
altered, can’t
bind substrate
Allosteric activator/inhibitor
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Substrate inhibition
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Inhibition of an enzyme activity by a substrate of the reaction
catalyzed by that enzyme; often, this type of inhibition occurs at
elevated substrate levels in which the substrate is binding to a second,
non-active site on the enzyme.
At high concentrations, some substrates also inhibit the enzyme
activity. Substrate inhibition occurs with about 20% of all known
enzymes. It happens when two molecules of substrate can bind to
the enzyme, and thus block activity.

Irreversible Inhibition
In irreversible
inhibition, the
inhibitor binds to the
enzyme irreversibly
through formation of
a covalent bond with
the enzyme ,
permanently
inactivating the
enzyme
Enzyme
S
O I
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Irreversible Inhibition - Reaction
Mechanism
In irreversible inhibition,
the inhibitor permanently
inactivates the enzyme.
The net effect is to remove
enzyme from the reaction.
Vmax decreases
No effect on Km
E + S ES E + P
EI
+
I
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The Michaelis-Menten plot for an
irreversible inhibitor looks like noncompetitive
inhibition
Vmax,app < Vmax
Km,app = Km
.
Vmax
Vmax2
1
2
1
Vmax,app
Km
Km,app
[Substrate]
ReactionRate
- Inhibitor
+ Inhibitor
Vmax,app
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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
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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.
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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
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Penicillin is a suicide inhibitor
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.
glycopeptide
transpeptidase
OHSer O
glycopeptide
transpeptidase
Ser
N
S CH3
CH3
COO-
H
H
C
C
O
H
N
C
O
H
H
R
N
S
CH3
COO-
H
H
C
C
O
H
N
C
O
H
Strained
peptide bond
Penicillin
CH3
R
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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.
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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)
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• 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
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Conclusion
There are several motivations to study enzyme
inhibition:
Distinguish among the different potential mechanisms in
multisubstrate reactions
Relative binding affinity of competitive inhibitors 
active site structure research in the absence of 3-D
structure info
Control mechanisms in biology: balance of protease and
their inhibitors in a tissue  help to achieve homeostasis
Commercial applications:
Insecticides, weed killers
Drugs

Since most clinical drug therapy is based on inhibiting the
activity of enzymes, analysis of enzyme inhibition kinetics is
fundamental to the modern design of pharmaceuticals
Well-known examples of such therapy include the use of
methotrexate in cancer chemotherapy to semi-selectively
inhibit DNA synthesis of malignant cells
the use of aspirin to inhibit the synthesis of prostaglandins
which are at least partly responsible for the aches and pains
of arthritis
the use of sulfa drugs to inhibit the folic acid synthesis that is
essential for the metabolism and growth of disease-causing
bacteria
In addition, many poisons (such as cyanide, carbon monoxide
and polychlorinated biphenols (PCBs)) produce their life-
threatening effects by means of enzyme inhibition .

References
BOOKS READ:
 Enzymes; Biochemistry, Biotechnology, Clinical chemistry
2nd edition
Page 126-150.
Trevor Palmer & Philip Bonner
East-West press private limited.
 LEHNINGER Principles of Biochemistry
5th edition
Page 183-233
Michael M.Cox & David L.Nelson
W.H.Freeman & company
WEBSITES VISITED:
 Search on www.google.com
 Search on www.youtube.com

Acknowledgement
I, Sandipayan Dutta, would like to express my heartfelt gratitude
towards our respected principal Dr. A . Nagarathna and our Head of the
Department Mrs. Asha.K.K .
I would like to give special thanks to Mrs. D.R. Jayashree for
assigning me the topic for presentation.
My family and friends have always been by my side and it falls as my
duty to mention them in this event.
rights reserved.

Sandipayan_enzyme inhibition_seminar

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