Inhibition of

Enzyme Activity
• Enzyme inhibitor is a substance that is capable of
diminishing velocity of the enzyme-catalyzed reaction.
• Two types of inhibitors are recognized: reversible and
irreversible.
• Inhibition by the reversible inhibitor can be
reversed, so that the enzyme activity is recovered.
• Inhibition by the irreversible inhibitor, on the other
hand, cannot be reversed.
•A. Reversible Inhibition
The reversible inhibitor (I) acts by forming a loose, dissociable complex (EI) with the
corresponding enzyme

•Catalytic activity of the complex (EI) is lower than that of the enzyme alone.
•the substrate transformation decreases after addition of the inhibitor.
•The reversible inhibition may be competitive, non-competitive, and uncompetitive.
•Following formation of the EI complex, the reaction may still obey Michaelis–Menten
kinetics but with altered Km and Vmax values that vary with the inhibitor concentration.
If the inhibitor acts only on the apparent Km, it is a competitive inhibitor;
if the inhibitor affects only the apparent Vmax, it is a non-competitive inhibitor;
and
if the inhibitor affects both the constants, it is an uncompetitive inhibitor
• Competitive Inhibition
Mechanism: The inhibitor exhibits is a close structural
resemblance with the actual substrate.
• It impairs substrate-binding to the enzyme, because it
occupies the active site of the enzyme making it unavailable
for the substrate.
• Occupancy of the enzyme active site by the inhibitor yields EI
complex.
• Since the formation of EI depends upon the concentration of
the inhibitor the inhibition is strictly dependent upon the
relative concentrations of the inhibitor and the substrate at
fixed concentration of the enzyme.
• Example:
Malonate ions inhibit the succinate dehydrogenase
reaction of Kreb’s cycle
• Effect on enzyme kinetics: The Km shows an apparent
increase in the presence of the inhibitor.
• This reflects decreased affinity of the substrate with the
enzyme active site.
• The decreased affinity is because the substrate must now
compete with the inhibitor for the active site.
• Vmax (the measure of fastest possible rate) remains
unchanged because the binding of inhibitor is reversible
• at infinite concentration of the substrate the inhibitor can
be completely displaced from the enzyme active site.
• Non-competitive Inhibition
Mechanism: the enzyme inhibitor is structurally unrelated to the structure of
substrate
• binds the enzyme at a site distinct from the enzyme active site.
• Occupancy of this site by the inhibitor alters the shape of
the enzyme such that its catalytic activity is reduced or lost.
• The substrate is still able to bind the enzyme, but the enzyme cannot catalyze
the reaction when inhibitor is bound.
• the non-competitive inhibitor does not block active site of the enzyme, but behaves
as though it were removing active enzyme from the solution.
• increasing the substrate concentration does not reverse the inhibition.
• The non-competitive inhibitor may bind the free enzyme as well as the enzyme
substrate complex, forming binary (EI) and ternary (ESI) complexes, respectively.
Both are catalytically inactive
 The effect of enzyme
inhibition
Examples
• Cyanide combines with the iron in the enzymes
cytochrome oxidase
• Heavy metals, Ag or Hg, combine with –SH groups.
These can be removed by using a chelating agent such as
EDTA.

© 2017 Paul Billiet ODWS

• Effect on enzyme kinetics: The non-competitive inhibitor
does not block the active site of the enzyme. Therefore, the
Km (indicating enzyme-substrate affinity) remains unaffected.
• Increasing the concentration of the substrate does not affect
binding of the inhibitor to the enzyme.
• This is because the shape of the enzyme is different when the
inhibitor is bound to it, and hence it does not matter how much
substrate is present; the enzyme will always be less effective
due to this shape change. This is reflected in decreased Vmax
• Uncompetitive Inhibition
The inhibitor binds only with the ES form of the enzyme to form the
enzyme-substrate-inhibitor (ESI) complex.
• Binding may occur at a site distant from the active site, or at a site
overlapping with the active site.
• result in lowering of both: the catalytic efficiency of the enzyme
and its apparent binding affinity for the substrate.
• Km and Vmax decreases).
• The uncompetitive inhibition is uncommon in single substrate
reactions.
• example in clinical enzymology is the inhibition of intestinal alkaline
phosphatase by L-phenylalanine.
•B. Irreversible Inhibition
The irreversible inhibitor binds covalently with an enzyme to form a stable complex.
•There is no dissociation of the enzyme and the inhibitor, so that the enzyme is
permanently inactivated.
•In some cases the inhibitor reacts at or near the active site of the enzyme with covalent
modification of the active site.
•In other cases, the inhibitor has no structural resemblance with the substrate and may
bind to a site other than the substrate-binding site. This type of inhibition resembles the
non-competitive inhibition, except that covalent bonds are formed in this case.
•The reaction of aspirin with cyclooxygenase represents an example of an irreversible
enzyme inhibition. Cyclooxygenase catalyzes the first reaction in the biosynthesis of
prostaglandin from arachidonate. Aspirin acetylates the serine residue at the active site;
the modification being highly stable leads to permanent loss of the enzyme activity.
Thus, aspirin is used as an antiinflammatory, antipyretic and anti-inflammatory drug.
• Suicide inhibition refers to irreversible enzyme inhibition
by a substrate analogue that, upon action of the enzyme,
generates highly reactive species.
• This species forms a covalent adduct with an amino acid side
chain at the active site, leading to irreversible inactivation of
the enzyme.
• An example of this type of inhibitor is allopurinol, the suicide
inhibitor of xanthine oxidase
• Therapeutic Applications of Enzyme Inhibitors
Several synthetic drugs (and natural compounds) exert pharmacological effects by acting as
enzyme inhibitors. This application is most easily appreciated with antiviral, antibacterial,
and antitumour drugs that are administered under the condition of limited toxicity. Those
drugs that resemble the substrate of an enzyme-catalyzed reaction are competitive inhibitors
with respect to that substrate (and non-competitive with respect to other substrates).
• Captopril, a commonly used drug in the treatment of hypertension
• competitively inhibit the angiotensin converting enzyme (ACE) which catalyzes the
proteolytic cleavage of angiotensin I to angiotensin II
• Angiotensin II elevates the arterial blood pressure.
• Captopril and other ACE inhibitors (e.g. lisinopril and enalapril) decrease
formation of angiotensin II, thereby lowering blood pressure (Table 6.8). The ACE inhibitors
contain a proline residue, which is an important substrate-binding determinant
• Lovastatin and mevinolin competitively inhibit activity of HMG-CoA reductase, the rate-
limiting enzyme in cholesterol biosynthesis. Therefore, they are commonly used
hypocholesterolaemic agents. Both the drugs resemble hydroxymethylglutaryl coenzyme A
(HMG-CoA), the natural substrate for this enzyme.
•Regulation of Enzyme Activity
There are three major mechanisms for regulating the
enzyme activity: allosteric modulation, covalent modification and induction-
repression of enzyme synthesis.
A. Allosteric Modulation
Some enzymes, called allosteric enzymes, exist in alternate higher order
structures.
•The equilibrium between these alternative structural forms can be affected by
binding of regulatory ligands, termed allosteric modulators.
•The binding of a ligand can either enhance the activity of
the enzyme (allosteric stimulation) or inhibit it (allosteric inhibition)
• The allosteric enzymes possess a site distinct and physically separate from the
substrate-binding site; it is known as the allosteric site (Greek word, allo which
means, other and stereos means space or site).
• Allosteric modulators bind with the allosteric site by reversible non-covalent interactions
. This binding alters conformation of the enzyme protein, which in turn leads to
alteration of the enzyme activity.
• occupancy of the allosteric site influences the enzyme activity, thereby providing important
means of its regulation.
• The process is relatively rapid.
• The allosteric site is specific for the modulator, just as the active site is for the substrate.
• The positive modulators increase the enzyme activity
• the negative modulators, decrease the enzyme activity.
• The allosteric site at which the positive modulator binds is referred to as an activator site;
the negative modulator binds at an inhibitory site.
• B. Covalent Modification
There are two general types of covalent modifications of
enzymes that regulate their activities.
• (a) reversible interconversion of active and inactive states of an enzyme
by covalent attachment of specific group(s)
• (b) irreversible activation of inactive enzyme precursor by proteolytic
cleavage.
Reversible Covalent Regulation
Activities of several enzymes are regulated by covalent
attachment of specific groups, such as phosphate, calcium or nucleotide.
This results in altered charged-state of the protein molecule, which in turn
causes change in shape of the protein and hence its activity.
• Irreversible Activation by Proteolytic Cleavage
Several proteins are synthesized in inactive forms.
• These are called zymogens, e.g. protein digesting enzymes and blood
clotting proteins.
• They are activated when a small length of the protein is cleaved off
from one end through action of specific proteases.
• This causes an irreversible rearrangement of the tertiary structure to
yield the active form of the protein.
• C. Induction-repression of Enzyme Synthesis
operates at gene-level to regulate the enzyme synthesis
(or breakdown).
• This may cause increased synthesis (induction) or
decreased synthesis (repression) of the enzyme protein
and hence its intracellular concentration also changes
accordingly.
• The sex hormones oestrogen and testosterone are two
very important regulators of genes.
• Insulin induces some key glycolytic enzymesand
represses some enzymes of gluconeogenesis
• Enzymes as Therapeutic Agents
• Enzymes find widespread use in treatment of a number of diseases:
• Streptokinase (from Streptococcus) or Urokinase (from urine) can cause lysis
of intravascular clots and therefore used in myocardial infarction.
• Hyaluronidase has degradative action against hyaluronic acid, a
mucopolysaccharide in extracellular tissue. Therefore, it is used for increasing
tissue permeability, and in treatment of traumatic or post-operative oedema.
• Asparginase is used as an anticancer drug; most commonly in some types of
leukaemia.
• Pepsin and trypsin are useful in patients with chronic indigestion and in
pancreatic insufficiency.
• Alpha-1-antitrypsin (AAT) is used in treatment of a type of emphysema that is
caused by deficiency ofAAT
• Collagenase degrades collagenous tissue, and so used
in management of severe burns and in debridement
of dermal ulcers.
• Lysozyme (endogenous antibiotic) is found in human
tears. It has antibacterial properties being active
against cellulose of bacteria, and is used in the
infection of eye.
• Penicillinase is a bacterial enzyme which degrades
penicillin. Therefore, it fi nds use in management of
patients who are allergic to penicillin.
• Papain is anti-inflammatory
• Streptodornase is a DNAase, applied locally.