SUICIDE INHIBITORS

Ms M. Mombeshora
Biochem Lecture 2
• Types of Inhibition
• Mechanism-based inhibition
• Suicide inhibitors
• Clinical examples
• Highlights
 Type of
Inhibitors

Reversible

Irreversible
Competitive

Active Site Uncompetitive

Directed

Suicide
Non- Competitive
Inhibitors
• An inhibitor is converted by the enzyme
catalytic mechanism to form an enzyme–
inhibitor complex
• Other terms used for mechanism-based
inhibitors include:
i. suicide inhibitors
ii. suicide substrate inhibitors
iii. alternate substrates
iv. substrate inhibitors
v. enzyme inactivators
vi. irreversible catalytic
inhibitors
• Suicide inhibition, also known as suicide
inactivation

• A form of irreversible enzyme inhibition

• Occurs when an enzyme binds a substrate

analogue and forms an irreversible complex
with it through a covalent bond during the
"normal" catalysis reaction
 Suicide inhibitors

• Inhibitor binds to active site where it is modified

by the enzyme

• It produces a reactive group that reacts

irreversibly to form a stable inhibitor-enzyme
complex

• Inhibitor has a functional group, usually a leaving

group, that is replaced by a nucleophile in the
enzyme active site
• Reaction with suicide inhibitor removes active
enzyme from the system; this removal is
measured as inhibition

• Is a relatively inert molecule that is transformed

by an enzyme at its active site into a reactive
compound that irreversibly inactivates the
enzyme

• Relatively unreactive until they bind to the active

site of the enzyme
E enzyme
SS suicide substrate
E.SS enzyme-suicide substrate complex
EX irreversible enzyme-suicide substrate complex
X activated intermediate
P product
E modified enzyme
 Suicide inhibitors

• First few steps of the reaction functions like a

normal substrate

• Then it is converted into a very reactive

compound that combines with the enzyme to
block its activity

• They use the normal enzyme reaction mechanism

to inactivate the enzyme
 Suicide inhibitors

• Suicide inhibitors that exploit the transition state-

stabilizing effect of the enzyme result in higher
enzyme binding affinity than do substrate-based
inhibitor designs

• Designing drugs that precisely mimic the transition

state is a real challenge because of the unstable,
poorly characterized structure of the transition
state
 Suicide inhibitors
• Prodrugs undergo one or more initial reactions
to form an overall electrostatic and three-
dimensional intermediate transition state
complex form with close similarity to that of the
substrate

• Serves as guidelines to further develop transition

state molecules with modifications

• Such inhibitors are called suicide inhibitors

because the enzyme appears to commit suicide
 Examples of Suicide inhibitors

 Allopurinol
• An anti-gout drug that inhibits xanthine oxidase
activity

• The enzyme commits suicide by initially

activating Allopurinol into Oxypurinol (a
transition state analog)

• The Oxypurinol binds very tightly to the

molybdenum-sulfide (Mo-S) complex at the
active site of xanthine oxidase
 Examples of Suicide inhibitors

 Acyclovir
• One of the most commonly used antiviral agents with very
low toxicity

• Is selectively converted into acyclo-guanosine

monophosphate (acyclo-GMP) by viral thymidine kinase

• Acyclo-GMP is further phosphorylated into the active

triphosphate form, acyclo-GTP, by cellular kinases
 Examples of Suicide inhibitors

• Acyclo-GTP is a very potent inhibitor of viral DNA

polymerase with over 100-fold greater affinity for
viral than cellular polymerase

• It is incorporated into viral DNA, resulting in chain

termination
 Examples of Suicide inhibitors

• The suicide inhibitor removes enzyme and reduces

the formation of ES complex

• The Vmax value is reduced and inhibition cannot be

overcome by adding extra substrate

• In this regard, suicide inhibition resembles

non-competitive inhibition
 5-fluorouracil
• Acts as a suicide inhibitor of thymidylate synthase
during the synthesis of thymine from uridine

• Reaction is crucial for proliferation of cells,

particularly those that are rapidly proliferating (e.g.
fast- growing cancer tumors)
During thymidylate synthesis, N5,N10- methyleneTHF is
converted to 7,8-dihydrofolate; methyleneTHF is regenerated in
two steps
 Examples of Suicide inhibitors

• Inhibition causes cell death from lack of thymine

to create more DNA

• This is often used in combination with Methotrexate,

a potent inhibitor of dihydrofolate reductase enzyme
 Examples of Suicide inhibitors
 Aspirin
• Inhibits cyclooxygenase

• Irreversibly inhibits COX-1 and modifies the

enzymatic activity of COX-2
 Examples of Suicide inhibitors

• Low doses of aspirin (75mg-81mg/day) sufficient to

irreversibly inhibit acetylate serine530 of COX-1

• Inhibits platelets generation of TXA2 –

antithrombin effect

• Intermediate doses (650mg-4g/day) inhibit COX-1 &

COX-2 blocking prostaglandins production
 Examples of Suicide inhibitors

 Penicillin
• Used medicinally as an antibiotic in the
treatment of many bacterial infections

• Derives its antibacterial action due to the fact

that it binds irreversibly to bacterial
transpeptidase
 Examples of Suicide inhibitors

• Inhibits DD-transpeptidase from building

bacterial cell walls

• Blocks the formation of the cross-link between

the N-acetylmuramic acid and N-
acetylglucosamine
• Special group of irreversible inhibitors relatively
unreactive until they bind to active site of enzyme

• Knowledge is highly useful in developing

pharmaceutical agents with minimal side effects

• Designing drugs that precisely mimic the

transition state - a real challenge because of
unstable, poorly characterized structure of
transition state
 Highlights
• Main goal of this approach - to create substrates
that are unreactive and non-toxic
• Become active within that enzyme's active site
and at the same time being highly specific for that
enzyme
• Drugs based on this approach have the advantage
of very few resulting side effects
• Suicide inhibitors are used in what is called
"rational drug design" where the aim is to create
a novel substrate, based on already known
mechanisms and substrates
Regulation of enzyme activity
 Regulation of enzyme activity

Regulate
• To control or direct according to a rule,
principle or law
Enzyme regulation
• The control of the rate of a reaction catalysed
by an enzyme and some effector such as
inhibitors or activators
• Or by alteration of some condition e.g pH or
ionic strength
 Importance of regulation of
enzyme activity

• Helps to use the substrate economically

• Regulates the metabolic pathways and their
interrelations
Ways by which enzyme activity
may be regulated
 Allosteric regulation
• Allosteric -occupying another space
• Substances which bind to allosteric site can
modify their activity – Allosteric effector

Allosteric effectors
• Substances that bind to allosteric site and
modifies the activity of the protein
 Allosteric regulation
• Binding of an allosteric effector induces
conformational change in the enzyme
• Allosteric activator and inhibitor exhibit positive
and negative cooperativities with the substrate
Allosteric activator :-
• On binding to the allosteric site promote binding
of substrate to the active site or the catalytic
action
• Allosteric inhibitor:- has opposite action
 Allosteric regulation
• Allosteric enzymes possessing more than one
substrate binding site on its subunits can bind
many substrates

• Binding of one substrate to active site increases

the affinity of other active site to substrate
Homotropic effect

• Allosteric modulator is different from the

substrate Heterotropic allosteric effect
 Allosteric regulation
• According to the model used (concerted
model) to study allosteric enzymes – they
exists in two conformational states:

• T (tense) and R (relaxed) state

 Mechanisms for Regulating
Enzyme Activity
1. Allosteric Enzymes
•Enzymes whose activity can be changed
by molecules (effector molecules) other
than substrate.
 Processes Involving the
Allosteric Enzyme
1. Negative Allosterism
- effector binding sites alters the shape
of the active site of the enzyme making
it to an inactive configuration.
2. Positive Allosterism
- effector binding sites that alters the shape
of inactive site of enzyme to an active
configuration.

Therefore, binding of the effector molecule

regulates enzyme activity by determining
whether it will be active or not.
 Feedback Inhibition
• An enzyme regulation process in
which formation of a product inhibits
an earlier reaction in the sequence
• It controls the allosteric enzymes

• This occurs when an end-product of a

pathway accumulates as the
metabolic demand for it declines
 Feedback Inhibition

• This end-product in turn binds to the

regulatory enzyme at the start of the
pathway and decreases its activity
• The greater the end-product levels
the greater the inhibition of enzyme
activity
 Feedback Control
• A product acts as a negative regulator
• When product concentration is high, it binds to an allosteric site
on the first enzyme (E1) in the sequence, and production is
stopped
• When product concentration is low, it dissociates from E1 and
production is resumed
• Feedback control allows products to be formed only when
needed
 Proenzymes (Zymogen)
• The inactive form of enzyme which
can be activated by removing a small
part on their polypeptide chain

• Mostly are the digestive enzymes and

blood clotting enzymes
 Proenzymes (Zymogen)

• Why is it that digestive enzymes are

in inactive state before it becomes
active?
• This is necessary to prevent digestion
of pancreatic and gastric tissues
 Active Form of
Zymogen Enzyme
• Pepsinogen Pepsin
• Trypsinogen Trypsin
• Prothrombin Thrombin
 Protein Modification
• Another mechanism that can turn on
and off the enzyme

• This a process in which a chemical

group is covalently added to removed
from the protein
• Phosphorylation, whereby a
phosphate is transferred from an
activated donor (usually ATP) to an
amino acid on the regulatory enzyme
• The most common example of type of
regulation
 Compartmentalisation

• Fatty acid synthesis takes place in cytosol

• Fatty acid oxidation takes place in

mitochondria

• Synthetic and catabolic pathways located in

different subcellular sites to achieve maximum
economy
 Induction and repression
• Affect the amount of enzyme present
• Increase in synthesis – Induction
• Decrease in synthesis – Repression

Inducer / Repressor
• Amount of enzyme directly controls the
velocity of the reaction
 Induction and repression
Constitutive enzymes :
• Level of which is fairly constant

Adaptive enzymes :
• Concentration increases or decreases as per the
need of the body
• Alteration in enzyme levels as a result of
induction and repression of enzyme protein
synthesis are slow (Hours to days)
 Induction and repression
Hormone insulin:
• Induces synthesis of – Glucokinase,
phosphofructokinase, Pyruvate kinase

• Represses Synthesis of – Pyruvate carboxylase,

Phosphoenol pyruvate carboxykinase (PEPCK),
Glucose 6 phosphatase
 Induction and repression
• Hormone Glucagon, Glucocorticoids and
epinephrine
• Induces synthesis of –
• Pyruvate carboxylase, Phosphoenol pyruvate
carboxykinase (PEPCK), Glucose 6 phosphatase

• Glucagon Represses Synthesis of –

• Glucokinase, phosphofructokinase, Pyruvate
kinase