Enzyme inhibition 1: reversible and irreversible enzyme inhibitors Flashcards
What is the general concept of enzyme inhibitors?
Enzyme inhibitors block the active centre and therefore modify the ability of the enzyme to catalyse a reaction with substrate.
What does enzyme inhibition lead to?
an increase of conc of substrate
Or a decrease in conc of the reaction product because the enzyme does not function properly
What are the modes of inhibition?
Inhibitors Possess high affinity/ability to bind than substrate
Reduce catalytic activity of enzyme or completely block it
What are the different classifications of inhibitors? (7)
Competitive reversible inhibitors Transition state inhib Non-competitive reversible (allosteric) Suicide inhibitors Substrate analogues Product mimics Non-competitve inhib – irreversible
How do reversible inhibitors bind?
binds and dissociates with the enzyme in an equilibrium process
How do irreversible inhibitors bind?
binds permanently through stable covalent bonds
Describe the complex formed by an irreversible inhibitor
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
Can irreversible inhibitors be displaced?
NO - therefore not competitive substrate is far too weak compared to the inhibitor
How does the enzyme recognise the inhibitor?
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
Examples of irreversible inhibitors?
nerve gases, penicillins, orlistat, disulfiram etc
How does a reversible inhibitor bind to the active centre?
Binds to active centre by sterically blocking it from the substrate. Enzyme is still effected though not permanently.
What does reversible inhibition depend on?
Depends on the strength of inhibitor binding and concentration
What reverses inhibition?
Increasing substrate concentration
How does reversible inhibition work?
Works by blocking the binding of the substrate to enzyme (steric hindrance) – obstructing catalytic reaction
Example of reversible inhibitors?
ACE inhibitors, statins, antidepressants, diuretics etc
What are competitive inhibitors?
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
What do competitive inhibitors do?
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
Does a reaction take place at the active site?
A competitive inhib may interact with enzyme at active site but no reaction takes place
Is competitive inhibition reversible?
Usually YES if sufficient substrate molecules are available to ultimately displace the inhibitor
What does amount of competitive inhibition depend on?
Inhibitor conc
Substrate conc
What are non-competitive inhibitors?
Chemical agent that interacts with the enzyme but usually NOT at the active site – usually allosteric site (SIDE OF THE ENZYME)
What do non-competitive inhibitors do?
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
Are non-competitive inhibitors reversible ?
usually yes
Are competitive inhibitors influenced by concentrations of the substrate?
NO - will never compete, increasing substrate conc will have no effect on inhibitor
What is the overall process of enzyme catalysis
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)
What does the enzyme have to go through?
High energy barrier through the transition step (mechanisms state inhibitor)
What is a ground state inhibitor?
Classification of inhibitor using mimicking
Involve substrate analogues
Product mimic
What are mechanism-based inhibitors?
Suicide inhibitors
Transition state analogues
Examples of reversible competitive inhibitors?
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
What is DHFR?
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
What are product analogues?
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
What is 6-mercaptopurine?
An allosteric inhibitor, prodrug used to treat leukaemia
What do serine, cysteine and histidine have?
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)
What are the different types of irreversible inhibitors?
Group-specific agents
Affinity labels
Suicide inhibitors
What are group-specific reagents?
Contain extremely active functional groups which can react with other functional groups – create a lot of side effects – not used in pharmaceutical practice
What are affinity labels ?
Quite reactive substrate analogues
Resemble chemical structure of substrate
Not always used as quite reactive
Covalently binds to active site
What are suicide inhibitors?
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
What is ORLISTAT?
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
What does the high reactivity of irreversible inhibitors mean?
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
