Enzyme inhibition 1: reversible and irreversible enzyme inhibitors

Question 1

What is the general concept of enzyme inhibitors?

Answer 1

Enzyme inhibitors block the active centre and therefore modify the ability of the enzyme to catalyse a reaction with substrate.

Question 2

What does enzyme inhibition lead to?

Answer 2

an increase of conc of substrate

Or a decrease in conc of the reaction product because the enzyme does not function properly

Question 3

What are the modes of inhibition?

Answer 3

Inhibitors Possess high affinity/ability to bind than substrate
Reduce catalytic activity of enzyme or completely block it

Question 4

What are the different classifications of inhibitors? (7)

Answer 4

Competitive reversible inhibitors 
Transition state inhib
Non-competitive reversible (allosteric)
Suicide inhibitors 
Substrate analogues
Product mimics
Non-competitve inhib – irreversible

Question 5

How do reversible inhibitors bind?

Answer 5

binds and dissociates with the enzyme in an equilibrium process

Question 6

How do irreversible inhibitors bind?

Answer 6

binds permanently through stable covalent bonds

Question 7

Describe the complex formed by an irreversible inhibitor

Answer 7

Form tight complex with enzyme via covalent bonds with certain amino acids in active site of enzyme
As a result, irreversible inhibitors may permanently suppress the catalytic activity of an enzyme
- known as INACTIVATORS

Question 8

Can irreversible inhibitors be displaced?

Answer 8

NO - therefore not competitive substrate is far too weak compared to the inhibitor

Question 9

How does the enzyme recognise the inhibitor?

Answer 9

Irreversible inhibitor shape mimics the substrate, so the enzyme recognises it and therefore binds to it. Followed by chemical reaction and is then covalently bound – totally INACTIVE, cannot be displaced

Question 10

Examples of irreversible inhibitors?

Answer 10

 nerve gases, penicillins, orlistat, disulfiram etc

Question 11

How does a reversible inhibitor bind to the active centre?

Answer 11

Binds to active centre by sterically blocking it from the substrate. Enzyme is still effected though not permanently.

Question 12

What does reversible inhibition depend on?

Answer 12

Depends on the strength of inhibitor binding and concentration

Question 13

What reverses inhibition?

Answer 13

Increasing substrate concentration

Question 14

How does reversible inhibition work?

Answer 14

Works by blocking the binding of the substrate to enzyme (steric hindrance) – obstructing catalytic reaction

Question 15

Example of reversible inhibitors?

Answer 15

ACE inhibitors, statins, antidepressants, diuretics etc

Question 16

What are competitive inhibitors?

Answer 16

Any inhibitor that will compete with a natural substrate to bind to active site of an enzyme
Must resemble chemical structure of natural substrate to be recognised by active centre

Question 17

What do competitive inhibitors do?

Answer 17

Prevents substrate molecules from reacting with enzyme and results in a decrease of reaction speed
However final product of reaction in UNCHANGED when the enzyme finally recognises the substrate

Question 18

Does a reaction take place at the active site?

Answer 18

A competitive inhib may interact with enzyme at active site but no reaction takes place

Question 19

Is competitive inhibition reversible?

Answer 19

Usually YES if sufficient substrate molecules are available to ultimately displace the inhibitor

Question 20

What does amount of competitive inhibition depend on?

Answer 20

Inhibitor conc

Substrate conc

Question 21

What are non-competitive inhibitors?

Answer 21

Chemical agent that interacts with the enzyme but usually NOT at the active site – usually allosteric site (SIDE OF THE ENZYME)

Question 22

What do non-competitive inhibitors do?

Answer 22

They change the 3D structure of the enzyme – shape of active centre changes – as a result the substrate may not recognise so will not bind or reduced ability to bind
-THEY DO NOT NEED TO MIMIC SUBSTRATE STRUCTURE

Question 23

Are non-competitive inhibitors reversible ?

Answer 23

usually yes

Question 24

Are competitive inhibitors influenced by concentrations of the substrate?

Answer 24

NO - will never compete, increasing substrate conc will have no effect on inhibitor

Question 25

What is the overall process of enzyme catalysis

Answer 25

Mimicking every step of the enzyme reaction
E + S –> ES complex at active site of the enzyme
ES then breaks down (either directly or through transition stage ES#)
ES# –> EP –> E+P (product mimics)

Question 26

What does the enzyme have to go through?

Answer 26

High energy barrier through the transition step (mechanisms state inhibitor)

Question 27

What is a ground state inhibitor?

Answer 27

Classification of inhibitor using mimicking
Involve substrate analogues
Product mimic

Question 28

What are mechanism-based inhibitors?

Answer 28

Suicide inhibitors

Transition state analogues

Question 29

Examples of reversible competitive inhibitors?

Answer 29

Methotrexate (anticancer agent) strong binding affinity
Pteridine ring binds flipped by 180 degrees but rest of molecule remains the same

Reaction catalysed by Dihydrofolate Reductase DHFR and Thymidylate synthase

Question 30

What is DHFR?

Answer 30

An enzyme crucial to maintaining the level of tetrahydrofolate (FH4), a methyl group shuttle required for the de novo synthesis of purines –> DHFR is an important pharmaceutical target

Catalyses the reduction of folic acid to FH4

Question 31

What are product analogues?

Answer 31

Normally represent reversible non-competitive inhibitors binding to the allosteric site
Final product binds to the allosteric site and switches off the enzyme
EXAMPLE  anti-purine, 6-methylthioinosine

This is an analogue that may block the first enzyme of the biochemical pathway
Its an allosteric inhibitor of the first enzyme in a biosynthesis pathway – represents the product minim

Question 32

What is 6-mercaptopurine?

Answer 32

An allosteric inhibitor, prodrug used to treat leukaemia

Question 33

What do serine, cysteine and histidine have?

Answer 33

NUCLEOPHILLIC residues (OH, SH and imidazole -groups)  can be attacked by ELECTROPHILLIC functional group (present in inhibitor) – haloketones (electronegative atoms pull the electrons from carbon atoms), diazoketone, epoxides, aziridine
Results in permanent damage / bonding
(IRREVERSIBLE)

Question 34

What are the different types of irreversible inhibitors?

Answer 34

Group-specific agents
Affinity labels
Suicide inhibitors

Question 35

What are group-specific reagents?

Answer 35

 Contain extremely active functional groups which can react with other functional groups – create a lot of side effects – not used in pharmaceutical practice

Question 36

What are affinity labels ?

Answer 36

 Quite reactive substrate analogues
 Resemble chemical structure of substrate
 Not always used as quite reactive
 Covalently binds to active site

Question 37

What are suicide inhibitors?

Answer 37

 Bind to enzyme as a substrate
 Can be processed by a normal catalytic mechanism leading to a generation of a chemically reactive intermediates that inactivate the enzyme through covalent modification

Question 38

What is ORLISTAT?

Answer 38

irreversible inhibitor of pancreatic lipase (lipase contains serine)
 Orlistat substrate comes along and lipase recognises it as a natural substrate which results in covalent binding so its blocked from digesting fats in the intestine. Less fatty acids and glycerol are absorbed as a result  USED AS ANTI-OBESITY DRUG – can be very harmful

Question 39

What does the high reactivity of irreversible inhibitors mean?

Answer 39

means that they are likely to be destroyed by hydrolysis, they can also interact with DNA (which can cause mutations and cancer eventually), and other molecules, creating a lot of side effects etc
Highly reactive so are wasted a lot of the time or destroyed, their lifetime action is also limited

Formation of glutathione and mercapturic acid conjugates