ENZYME INDUCTION AND

INHIBITION
Inhibitors and Inducers
• The rates of enzymatic reactions are often affected by
substances other than the enzyme or substrate.
• These modifiers may be inhibitors because their
presence reduces the reaction rate
• or activators because they increase the rate of
reaction.
• Activators and inhibitors are usually small molecules
(compared with the enzyme itself) or even ions.
• They vary in specificity from modifiers that exert
similar effects on a wide range of different enzymatic
reactions at one extreme, to substances that affect
only a single reaction
• Enzyme is a biological catalyst, i.e. a substance that alters
the rate of a reaction without itself becoming permanently
altered by its participation in the reaction.
• The ability of an enzyme (particularly a proteinaceous
enzyme) to catalyze a reaction can be altered by binding
various small molecules to it, sometimes at its active site
and sometimes at a site distant from the active site.
• Usually these alterations involve a reduction in the
enzyme's ability to accelerate the reaction; less commonly,
they give rise to an increase in the enzyme's ability to
accelerate a reaction.
 Inhibitors:

• Inhibitors are chemicals that reduce the rate of

enzymatic reactions,
• They are usually specific and they work at low
concentrations,
• They block the enzymatic action but they do
not usually destroy them.
• Inhibitors are broadly classified as reversible and irreversible.
• Irreversible inhibitors bind permanently to their target
enzyme, often via a covalent bond that influences catalysis
• For example, organophosphorous compounds are extremely
potent irreversible inhibitors of esterases, including
acetylcholinesterase
• An irreversible inhibitor combines covalently with the
enzyme so that physical methods are ineffective in separating
the two
• Irreversible inhibition can be rarely seen i.e, they are not as
common as Reversible inhibition.
• Inhibitors can be washed off the body by dialysis, gel
filtration or chromatography process.
 Type of Enzyme Inhibitors

Reversible
• Competitive
• Uncompetitive
• Non- Competitive
Irreversible
• Active Site Directed
• Suicide / kcat Inhibitors
 Reversible Inhibition

• Reversible inhibition implies that the activity of

the enzyme is fully restored when the inhibitor is
removed from the system in which the enzyme
acts by some physical separative process,
• such as dialysis, gel filtration or chromatography.
 Reversible Inhibition

• Inhibitor binds to Enzyme reversibly through weak non-

covelent interactions
• An Equilibrium is established between the free inhibitor and
EI Complex and is defined by an equilibrium constant (Ki)
E+I EI

• The activity of Enzyme Is fully restored on removing the

Inhibitor by dialysis.
• Reversible Inhibitors depending on concentration of E, S
and I, show a definite degree of inhibition which is reached
fairly rapidly and remains constant when initial velocity
studies are carried out.
 Competitive Inhibition

• A competitive I combines with the free enzyme

to form an EI complex in a manner that
prevents S binding
• Binding of S and I is mutually exclusive
• Inhibition can be reversed by increasing the
concentration of S at a constant [I]
• Degree of inhibition will depend on the
concentrations of S and I and on the relative
affinities of the enzyme for S and I
 Non-competitive Inhibition

• An inhibitor that binds to an enzyme to form a dead end

complex, whether or not the active site is occupied by a
substrate is termed as a NC Inhibitor
• Can bind either to E or ES complex
• Since I doesn't bear structural resemblance to the S, it must
bind to the enzyme at a site distinct from the S binding site
• The presence of I does not affect S bonding but does
interfere with the catalytic functioning of the enzyme
• The binding of I often deforms the E so that it doesn’t form
ES complex at a normal rate and once formed, ES complex
doesn’t decompose at normal rate to yield products
• A NC-I doesn’t affect the Km because the
binding of I does not block S binding or vice-
versa
• I effectively lowers the concentration of active
enzyme and hence decreases the apparent
Vmax
• since there is no competition between S & I,
the inhibition is not reversed by increasing the
[S]
• Examples for Non- Competitive Inhibition
• 1. Enzymes requiring divalent metal ions (e.g. Mg2+
& Ca2+ etc) for their activity are inhibited non-
competitively by chelating agents like EDTA which
removes metal ions from the enzyme
• 2. Enzymes with -SH groups that participate in the
maintenance of the three dimensional conformation
of the molecule are non-competitively inhibited by
heavy metal ions.
• E SH + Hg2+ E S Hg+ + H+
 Uncompetitive Inhibition

• I doesn't bind to the free E rather it binds to the ES complex

the binding of an UC I is presumed to cause structural
distortion of the active site making the enzyme catalytically
inactive
• the binding of S could cause a conformational change in the
E thereby revealing an I binding site
• Inhibition can’t be reversed by increasing the [S] since I
doesn't compete with S for the same binding site
• UC Inhibition is rare in single-substrate reactions
• for e.g. Inhibition of intestinal alkaline phosphatase by L-
phenylalanine
• It is common in multisubstrate reactions
 Irreversible Inhibition

• Inhibitor binds at or near the active site of the

enzyme irreversibly, usually by covalent bonds, so it
can’t dissociate from the enzyme
• No equilibrium exits
E+I EI

• Enzyme activity is not regained on dialysis

• Effectiveness of I is expressed not by equilibrium
constant but by a velocity constant, which determines
the fraction of the enzyme inhibited in a given period
of time by a certain concentration of the I
 Irreversible Inhibition

• An irreversible Inhibitor binds at or near the active site

of the enzyme irreversibly, usually by covalent bonds, so
that it can’t subsequently dissociate from the enzyme
• The I destroys as essential functional group on the
enzyme that participates in normal S binding or catalytic
action.
• As a result the enzyme is rendered permanently inactive
• Compounds which irreversibly denature the enzyme
protein or cause non-specific inactivation of the active
site are not usually regarded as irreversible inhibitors.
 Irreversible inhibitors

Types of Irreversible Inhibitors

Suicide
Active site Inhibitors
directed (Mechanism-
irreversible based Inhibitors)
Inhibitors or
(kcat Inhibitors)
or
(Affinity labels)
 Affinity labels

• An affinity label is a chemically reactive

compound that is designed to resemble the
substrate of an enzyme so that it binds at the
active site and forms a stable covalent bond
with a susceptible group of the nearby residue
in the enzyme protein.
• Affinity labels are very useful for identifying
catalytically important residues
 Suicide Inhibitors

• A suicide inhibitor is a relatively inert molecule that is

transformed by an enzyme at its active site into a reactive
compound that irreversibly inactivates the enzyme
• They are substrate analogs designed so that via normal
catalytic action of the enzyme, a very reactive group is
generated.
• The latter forms a covalent bond with a nearby functional
group within the active site of the enzyme causing
irreversible inhibition.
• Such inhibitors are called suicide inhibitors because the
enzyme appears to commit suicide.
• e.g. FdUMP is a suicide inhibitor of thymidylate synthase.
 Suicide inhibition

• It is a type of irreversible inhibition.

• Also known as mechanism based inactivation.
• Here , the structural analogue is converted into
a more effective inhibitor with the help of the
enzyme to be inhibited.
• The substrate like compound initially binds
with the enzyme and the first few steps of the
pathway are catalysed.
• This new product irreversibly binds to the
enzyme and inhibits further reactions.
 Alloprinol
• Substrate analogue of Hypoxanthine
• 1.Hypoxanthine
• XO (xanthine oxidase)
•
• xanthine
• xanthine oxidase
• xanthine oxidase inhibits
• Uric acid 1. Allopurinol alloxanthine
• 2. 5fluorouracil is used in the treatment of
cancer
• 5Fluorouracil 5fluoro-deoxyuridylate
•
• dUMP TMP
• Thymidylate synthase
 Importance of Enzyme Inhibition

• For understanding the regulation of enzyme activity within the living cells
• To elucidate the kinetic mechanism of an enzyme catalyzing a multi-
substrate reaction
• Useful in elucidating the cellular metabolic pathways by causing
accumulation of intermediates
• Identification of the catalytic groups at the active site
• Provide information about substrate specificity of the enzyme
• Form the basis of drug designing.
• The whole area of selective toxicity , including the use of antibiotic,
toxin, insecticides etc is based on the exploitation of species differences in
the susceptibility to enzyme inhibitors.
• Competitive inhibitors are useful in x-rays crystallographic studies to pin
point the active site in crystal structure and thus revealing how the
surrounding amino acid residues interact with the bound molecule.
• To treat methanol poisoning
 Rx of methanol poisoning

• Ethanol: analogue of methanol, used for the

treatment of methanol intoxication.
• CH3OH AD HCHO (formaldehyde)

HCOOH (Formic acid)

• HCHO Causes retinal damage & blindness

• HCOOH produces severe acidosis & death
 Methanol inhibition

• CH3CH2OH CH3CHO CH3COOH

• NAD NADH
• CH3CHO Less toxic

• Ethanol competes for alcohol dehydrogenase

& prevents methanol toxicity.
 Inducers/activators
• The process of increasing the amount or the activity of a
protein – Enzyme induction.
• A homeostatic mechanism for regulating enzyme
production in a barrier organ, such as the liver, intestine,
kidney.
• The inducer usually combines with and
deactivates/activates a regulatory protein which leads to
increased gene expression.
• Many of the enzymes involved in drug metabolism may be
up-regulated by exposure to drugs and environmental
chemicals leading to increased rates of metabolism.
 REGULATION OF ENZYME ACTIVITY

• 1. Compartmentalization of Pathways
• 2. Induction and Repression of Enzymes
• 3. Degradation of Enzymes
• 4. Covalent Regulation
• 5. Allosteric Regulation
• 6. Iso enzymes
1. Compartmentalization
• Enzymes for fatty acid synthesis are present in cytosol
• Enzymes for oxidation of fatty acids are present in
mitochondria.
2. Induction & Repression
• Induction is increased synthesis of enzymes at gene level
through hormones or other substances.
• Eg: Insulin induces the synthesis of glucokinase /glycogen
synthase.
• Repression is decreased synthesis of enzymes at gene level.
• Eg: Ala synthase is repressed by heme.
• 3. Enzyme degradation
• The regulatory /key enzymes are degraded if
not needed and they are rapidly synthesized
when required.
• Half lives of enzymes vary from one another.
4.Covalent Regulation
• 1.Irriversible by hydrolysis of chemical bonds -
proenzymes.
• eg: Inactive enzymes Active enzymes
Zymogen / Proenzyme
• Pepsinogen HCL Pepsin
• 2. Reversible by phosphorylation and
• dephospharylation of enzymes
• eg: Glycogen phosphorylase ( a & b)
 5.Allosteric Inhibition:-

• 1.In glycolysis
• Glucose Hexokinase glucose-6phosphate
ATP ADP
• Glucose-6phosphate acts as allosteric inhibitor for
hexokinase
• 2.F-6phosphate Phospho fructo kinase F-1,6bisphosphate
ATP ADP
• Acetyl CoA Acetyl CoA carboxylase Malanoyl CoA

Palmitate

• Palmitate acts as negative allosteric modulator

for acetyl CoA caboxylase