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Enzyme inhibition and
regulation
Ms Lulembo

1

•

Enzyme inhibitors are molecular agents that interfere with catalysis, slowing
or halting enzymatic reactions
• Inhibitors are the most important pharmacological agents known
Enzyme inhibitors – have provided valuable information about enzyme
mechanisms
- have helped define some metabolic pathways
Two broad classes – Reversible and irreversible
Reversible inhibition
Can be – competitive, uncompetitive, noncompetitive or mixed
A competitive inhibitor (I)
• competes with the substrate for the active site of the enzyme
• while the inhibitor occupies the active site it prevents binding of the
substrate to the enzyme
• competitive inhibitors often resemble the substrate and combine with the
enzyme to form an EI complex, but without leading to catalysis
• thus a competitive inhibitor diminishes the rate of catalysis by reducing the
proportion of enzyme molecules bound to a substrate
2

E

+ S

ES

E+P

+
I

EI
•

•

•

The I is bound reversibly to the enzyme and when more substrate is added
the inhibition is overcome by pulling the enzyme free via breakdown of EI
complex which is in equilibrium with free E and I
...

1
...
Methotraxate
• Is a potent competitive inhibitor of dihydrofolate reductase, an enzyme
which plays a role in the biosynthesis of purines and pyrimidines
• It is a structural analogue of dihydrofolate, a substrate for dihydrofolate
reductase
• It binds to the enzyme 1000-fold more tightly than the natural substrate and
inhibits nucleotide base synthesis
• Its used to treat cancer
Uncompetitive inhibition
• The inhibitor binds only to the enzyme-substrate complex
E + S

ES + I

E+P

Ki
ESI
•
•
•

Because some unproductive ESI are always present, the Vmax is lower in the
presence of the inhibitor than in its absence
The uncompetitive inhibitor lowers the apparent Km
...
To maintain
equilibrium between E and ES more S binds to E
5

•

It can not be overcome by addition of more substrate

Non-competitive inhibition which is also reversible,
• The inhibitor and substrate can bind simultaneously to an enzyme molecule and
binding sites do not overlap
• A noncompetitive inhibitor acts by decreasing the turnover number rather than by
diminishing the proportion of enzyme molecules that are
bound to substrate
• Can not be overcome by increasing substrate concentration

E + I
Ki

EI
•
•

S

S

ES + I

E+P

Ki
ESI

X

The Vmax is decreased to a new apparent Vmax whereas the Km remains unchanged
The inhibitor simply lowers the concentration of functional enzyme; behaves like a
more dilute solution of enzyme
• If Ki for I binding to empty enzyme E is not the same as that for I binding to occupied
ES, mixed inhibition may be observed
In mixed inhibition
• a single inhibitor hinders both the binding of substrate and decreases the turnover
number of enzyme
6

Irreversible inhibitor
• An irreversible inhibitor dissociates very slowly from its target enzyme because it
becomes very tightly bound to the enzyme, either covalently or noncovalently
eg -action of nerve gas on acetylcholinesterase, an enzyme that plays an
important role in the transmission of nerve impulses
-heavy metal ions (Ag+, Hg 2+)
-iodoacetamide
•

Some irreversible inhibitors are important drugs e
...

– Penicillin acts by covalently modifying the enzyme transpeptidase, preventing the
synthesis of bacterial cell walls thus killing the bacteria
– Aspirin acts by covalently modifying the enzyme cyclooxygenase, reducing the
synthesis of signaling molecules involved in inflamation

Suicide inactivators (mechanism designed inactivators) – are a special
class of irreversible inhibitors
• A suicide inactivator carries out the first few chemical steps of the
normal enzyme reaction, but instead of being transformed into the
normal product, the inactivator is converted to a very reactive
compound that combines irreversibly with the enzyme
eg Allopurinol, a drug used for treatment of gout first acts as a
substrate and then as an inhibitor of the enzyme xanthine oxidase

7

ENZYME REGULATION
• Not all enzymes are required to work all the
time
...

• There are 4main enzyme regulation
mechanisms:
• -use of effectors
• -allosteric regulation
• -covalent regulation
• -regulation by hormones
8

Regulatory Enzymes
• Each pathway has one or more enzymes with greater
effect on overall metabolic sequence
• These regulatory enzymes exhibit an increase or
decrease in catalytic activity in response to certain
signals
• Adjustments in the rate of reactions catalyzed by
regulatory enzymes and therefore the rate of entire
metabolic process allow cells to meet changing needs
for energy and biosynthesis
• In each metabolic pathway the enzyme that catalyzes
the slowest or rate limiting step sets the rate of overall
sequence
• In most multienzyme systems the first enzyme of the
sequence is the regulatory enzyme – catalyzes the
committed step
9

Use of effectors
• Enzyme synthesis is controlled at the level of
DNA synthesis
• 2 types of effectors- inducers and repressors
• When an enz is not required a repressor binds
to the operator on DNA and transcription is
blocked
• When an enzyme is required the inducer binds
to the repressor and the operator is free so
transcription proceeds
10

Features of allosteric enzymes
• Undergo a conformational change in response to modulator binding
• Are generally larger than non allosteric enzymes with two or more
subunits
• Modulator may be inhibitory or stimulatory
• Often the substrate itself is the modulator(homotropic regulation)
• Binding of substrate causes a conformational change that affect
subsequent activity on other sites
• When the modulator is a molecule other than substrate its referred
to as heterotropic regulation
• Allosteric modulators should not be confused with inhibitors
• In addition to active sites, allosteric enzymes generally have one or
more regulatory or allosteric sites for binding the modulator
• In homotropic regulation, the active site and the regulatory site are
usually the same
• In many allosteric enzymes the substrate binding site and the
modulator binding site(s) are on different subunits; the catalytic (C)
and the regulatory (R) subunits
– Modulator binds to a specific site on regulatory subunit

11

• Allosteric enzymes bind activators at the allosteric site, a
site physically separate from the catalytic site
...
Generally at the earliest
functionally irreversible step unique to a particular pathway
– The regulatory enzyme activity slows; subsequent enzyme rates
operate at different rates as their substrates are depleted
– Rate of end product production is brought in line with cell’s
needs
– Called feedback inhibition
– Eg aspartate transcarbamoylase, which catalyzes the first
reaction unique to pyrimidine biosynthesis is feedback inhibited
by cytidine triphosphate
– In simple product inhibition the immediate product of enzyme
activity inhibits the enzyme

13

Some regulatory enzymes undergo reversible covalent modification
• Modifying groups include phosphoryl, adenylyl, uridylyl, adenosine
diphosphate ribosyl and methyl groups
• These groups are generally covalently linked to and removed from the
regulatory enzymes by separate enzymes
• Phosphorylations make up the vast majority of known regulatory
modifications
• Some proteins have one phosphorylated residues, others have
many

14

Covalent modification
Phosphorylation

Amino acid residues known to accept
covalent modification
Tyr, Ser, Thr, His

Adenylylation

Tyr

Uridylylation

Tyr

ADP-ribosylation

Arg, Gln, Cys

Methylation

Glu

15

γ
O
─
O

P
O─

α

β

O

O

P
─
O

Adenine

O

P
O

O

O

CH2

─

H

H

OH

H
OH

H

The terminal (γ) phosphoryl
group is transferred to
hydroxyl groups of specific
serine threonine and
tyrosine

Adenosine triphosphate (ATP)

16

• Nerve impulses—and binding of hormones to
cell surface receptors—elicit changes in the rate
of enzyme catalyzed reactions within target cells
by inducing the release or synthesis of
specialized allosteric effectors called second
messengers
...

17

Some types of regulation require proteolytic cleavage of an enzyme
precursor
• Certain proteins are synthesized and secreted as inactive
precursor proteins known as proproteins
• An active precursor – a zymogen is cleaved to form the active enzyme
-Many proteolytic enzymes (proteases) of the stomach and pancreas
are regulated in this way
-Chymotrypsin and trypsin are synthesized as chymotrypsinogen and
trypsinogen
-Specific cleavage causes conformational changes that expose the enzyme
active sites
-Because activation is irreversible – inactivated by other mechanisms
• Proteases are inactivated by inhibitor proteins that bind very tightly
to the enzyme inhibitor site
eg pancreatic trypsin inhibitor binds to and inhibits trypsin
• Proenzymes eg clotting system
Fibrin is produced by proteolysis of fibrinogen

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