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Flashcards in Medicine -Enzyme Inhibitors 1 Deck (53):

What is kcat?

Overall rate constant for decomposition of enzyme-substrate complex into products


How to reversible enzyme inhibitors work?

Reversible inhibitors find reversibly to the active site via intermolecular interactions with the enzyme.
The inhibitor blocks access for the substrate to the enzyme active site. The inhibitor undergoes no reaction in the enzyme active site


Name some pharmaceutical drawbacks of irreversible inhibitors

-their inherent chemical reactivity: they are designed to be alkylting or acylating agents, thus they can be wasted, destroyed or attacked by other cellular components with unpredictable consquences
-their effective lifetimes are also limited by the rate of protein turnover for the target enzyme (i.e. New protein wil be biosynthesised and perhaps induced) in constant, reversibility bound drug can be released as its protein target gets turned over by proteolysis etc. It can then be re-used at new target sites of another protein molecules


Examples of irreversible inhibitors

Nerve gases
Protein Pump Inhibitors (PPIs)


What is glutathione?

Glutathione (GSH) is a tripeptide which protects cells from toxins


Name two types of mechanism-based enzyme inhibitors

Transition state mimics
Suicide or kcat inactivators


Name 2 modes of enzyme inhibition (and result of it)

-decrease in catalytic activity of an enzyme
-decrease in the ability of an enzyme to bind its substrate
Or both of the above


What is the general concept of enzyme inhibition?

Interact in a prohibitive way with an enzyme (e.g. Block the enzyme active site)
Modify the ability of the enzyme to catalyse the reaction of its natural substrates
Prevent enzyme from working in its normal manner


How are enzyme inhibitors classified broadly?

In many ways:
-based on the modes of their interaction with the enzyme: i.e. Reversible: bind and dissociate with enzyme in an equilibrium process
Or irreversible: bind tightly and form an essentially permanent complex via covalent bonds
-by considering the emulation of the certain step of an enzymatic reaction


How and where do transition state analogues work?

Structurally resemble the substrate portion of the unstable transition state (ES*) of an enzymatic reaction

Act at highest energy of graph (mechanism-based inhibitor)


How do suicide inhibitors react? Where?

Bind to the enzyme as a substrate
Can be processes by a normal catalytic mechanism leading to the generation of a chemically reactive intermediate(s) that inactivate the enzyme through covalent modification


What is 5-fluorouracil Reltitrexed and what does it do?

Inhibits the enzyme thymidylate synthase

Thymidylate synthase catalyses the reaction of

dTMP is a DNA building block needed for DNA synthesis and cell division

Hence, 5-fluorouracil inhibits DNA synthesis and cell division


Structure of competitive inhibitor in relation to the substrate?

A competitive inhibitor is a compound which closely resembles the chemical structure and molecular geometry of a substrate


What does the effect of increasing substrate concentration do to irreversible inhibitors?

Increasing substrate concentration does not reverse the inhibition of irreversible enzyme inhibitors


What does an increase of substrate concentration do when you have a reversible inhibitor near an enzyme?

An increase in substrate concentration reverses the inhibition


How do affinity labels react? What are they?

Reactive substrate analogues, structurally similar to the substrate
Capable of binding to the active site
More specific than group-specific reagents


Name 5 types of electrophilic chemical groups used for covalent labels
State the two which are the least electrophilic

-Sulfonyl halide
-Alpha-haloketones (Cl, Br, I)
Less electrophilic:


Reversible competitive inhibitor action (good and bad)

-competes with a substrate for access to the enzyme active site
-this prevents the substrate molecules from reacting with the enzyme and results in a decrease of the observed reaction velocity
-The competitive inhibitor leaves the actual end product unchanged
-A competitive inhibitor may interact with the enzyme at the active site, but have no reaction taking place.


How do product analogues work?

Enzyme with allosteric sites is controlled by the final product of the biosynthetic pathway
Final product binds to the allosteric site and switches off the enzyme
Product analogue can be used in the design of enzyme inhibitors


Name 3 amino acids that possess nucleophilic residues
Name the residues

Serine (-OH)
cysteine (-SH)
histidine (imidazole)


The amount of inhibition of a reversible competitive inhibitor depends on

-the inhibitor concentration
-the substrate concentration
-relative affinities of the inhibitor and substrate for the active site


What does inhibition of a suitably selected target enzyme lead to?

-an increase in the concentration of substrate
-a decease in the concentration of the product (or metabolite)
One or both of the above leads to the required clinical response


Describe the overall process of enzymatic catalysis

-Enzymes (E) catalyse the reactions of the substrate(s) by initial formulation of a complex (ES) at the active site of the enzyme
-ES breaks down, either directly or via an intermediate stage (e.g.transition state ES*)
-This results in formation of the product (P) followed by regeneration o the enzyme to give E


How do non-competitive reversible enzyme inhibitors act?

-The non-competitive inhibitor reacts either distant from or adjacent to the active site, i.e. At the allosteric site
-It changes the 3D structure of the enzyme, subsequent change of the shape of the enzyme active site
-this reduces the ability of the enzyme to recognise and interact with substrate and thus decrease the catalysis of the enzymatic reaction


Dihydrofolate reductase is what?

An enzyme crucial to maintaining the level of FH4, a methyl group shuttle required for the de novo synthesis of purine
DHFR is an important pharmaceutical target
Catalyses the following two reactions i.e. Catalyses the reduction of folic acid to FH4 in two steps:
Folic acid→FH2→FH4


How do group specific reagents work? Give 2 examples

React with specific amino acid side chains, e.g. DIPF or iodoacetamide


How does anti-purine 6-methylthioninosine work?

It is the end product of purine biosyntheis
It is an allosteric inhibitor of an earlier enzyme in the purine biosynthesis pathway


Methotrexate is what?

An anti-cancer drug that inhibits DHFR


Name and describe the drawbacks of suicide inhibitors

-Inhibitor structure is specific but may still bind to other cell components
-substrate recognition may be shared at a binding level, even if other protein doesn't activate the chemical reaction
-there is a problem if the target enzyme is synthesised in the cell as an inactive pre-cursor
-in some cases suicide inhibitors can cause toxicity due to their high reactivity (e.g. Tienilic acid)


What is bad about the high reactivity of irreversible inhibitors?

It means that they are likely to be destroyed (wasted) by hydrolysis or be de-toxified by nucleophiles such as GSH (glutathione) and glutathione S-transferases:
-by direct reaction
-or by enzyme catalysed reactions


Show a biosynthetic pathway with feedback control


P4 then inhibits enzyme that catalyses S⇄P1


How and where do product mimics work?

Emulate structure of the product, unbound or bound to enzyme

Lowest energy (ground state inhibitors)


Influence of concentrations of the substrate on non-competitive reversible inhibitors

Not influenced by concentrations of the substrate


Name 3 types of irreversible (non-competitive) inhibitors

Suicide inhibitors
Affinity labels
Group-specific reagents


Overall process of enzyme catalysis with letters

E + S ⇄ ES ⇄ES*⇄EP⇄(kcat)⇄E + P


Where do irreversible inhibitors act?

May act at the active site or remote from it. Consequently, they may not be displaced by the addition of excess substrate because in any case, the basic structure of the enzyme is modified to such a degree that it ceases to work


Possible mechanisms for reversible inhibitors?

-blocking binding of the substrate to the enzyme
-obstructing catalytic reaction (or both)


What is Orlistat?

-Orlistat is an anti-obesity drug that inhibits pancreatic lipase.
-The enzyme is blocked from digesting fasts in the intestine
-Fewer fatty acids and glycerol are absorbed as a result
-This leads to reduced biosynthesis of fat in the body


How can mercapturic acids be formed by electrophilic functional groups?

Electrophilic functional groups (epoxides, alkyl halides, sulfonates, dilsulfides, radical species etc) can react with nucleophilic thiol group of glutathione to produce glutathione conjugates, which can then be transformed to mercapturic acids


Describe how the shapes of irreversible and reversible enzyme inhibitors are different, if at all

Reversible: Inhibitor likely to be similar in structure to the substrate (e.g. Reversible substrate analogue or reversible product mimic)

Irreversible: likely to be similar in structure to the substrate but doesn't have to be


How do irreversible (non-competitive) inhibitors work?

Inhibitor binds irreversibly to the active site
Covalent bonds (with certain amino acids in the active site of the enzyme) formed between the inhibitor and the enzyme, forming a tight complex
Substrate is blocked from the active site
As a result, irreversible inhibitors may permanently suppress the catalytic activity of an enzyme
Also termed inactivators


Give 2 examples of product analogues/natural products that alter biosynthetic pathways

Anti-purine, 6-methylthioinosine


The most effective irreversible inhibitors have what properties?

Those that can react with an amino acid in the active site to form covalent bond(s)


Name three types of ground state inhibitors

Product mimics

Substrate analogues
Affinity labels


What is the difference between methotrexate and FH2?

Methotrexate (potent drug) is a structural mimic of FH2
The only two small changes are:

C=O vs NH2
H vs CH3

Methotrexate has stronger binding affinity for DHFR due to the additional hydrogen bond, which is not present when FH2 binds


What does the inhibition of a reversible inhibitor depend on?

-strength of inhibitor binding
-inhibitor concentration


What is a non-competitive reversible enzyme inhibitor?

A chemical agent that interacts with the enzyme, but not at the active site


Name types of reversible enzyme inhibitors

-allosteric inhibitors
-feedback-based inhibitors


How and where do substrate analogues work?

emulate the structure of unbound or bound substate at its ground state

Start of reaction (E+S)


Examples of reversible inhibitors

ACE inhibitors
Protease inhibitors
Kinase inhibitors


How does 6-Mercaptopurine work? What is it?

Treats leukaemia
Prodrug which is converted to its corresponding nucleoside monophosphate by cellular enzymes
Allosterically inhibits the first enzyme in biosynthesis of purines
Blocks the biosynthesis of purines and DNA


Categorise enzyme inhibitors into two main groups



What does methotrexate end up doing in the long run?

It inhibits production of FH4, and consequently, inhibits production of N5,N10-methylene-FH4 which is involved in the methylation of dUMP to form dTMP
Without FH4, the synthesis of dTMP (the DNA building block) is blocked, which results in the inhibition of DNA synthesis