Enzyme InhibitionDr.Geeta Jaiswal

InhibitorsEnzymes are proteins. They

can be inactivated by agents which denature them.

Chemical substances that inactivate enzymes are called Inhibitors

Inhibitors are organic substances or inorganic ions

Inhibitors decrease enzyme activity without altering the ionic strength or pH of the medium

Inhibition may be reversible , where the inhibitor does not react covalently with the enzyme.

Some agents react covalently with the functional groups of enzymes resulting in non-competitive irreversible inhibition.

Inhibition

Reversible Irreversible

Competitive Non-Competitive Uncompetitive

Classification of Inhibition

Competitive Inhibition

It is called substrate analogue inhibition

Inhibitors resembles substrate and competes for the active site of the enzyme.

Inhibitor binds with enzyme to form EI complex .

Due to structural similarity the substrate and inhibitor molecule both compete for the active site

This is a reversible type of inhibition, where both ES and EI complexes are formed.

The actual amount of ES and EI will depend upon:- (i) Affinity between enzyme

substrate/and inhibitor present. (ii)Actual concentration of

substrate and inhibitor present (iii) Time of pre-incubation with

substrate or inhibitor

Line weaver’s Burk plot representing Competitive Inhibition

Competitive Inhibition

Affinity Decreases

I Decreases as Km increases

Km

I Remains the same

Vmax

Efficiency Remains the same

Examples of Competitive Inhibitors

S.N Enzyme Substrate Inhibitor 1. LDH Lactate Oxamate

2. Aconitase Cis-Aconitase Trans-Aconitase

3. Succinate Dehydrogenase

Succinate Malonate

4. H.M.G Co.A reductase HMG Co.A HMG

5. Di Hydrofolate Reductase

7,8 dihydrofolate Amethopterin

6. Xanthine Oxidase Hypoxanthine Allopurinol used in Gout Treatment

Examples of Competitive Inhibitors used as Drugs Clinically

Allopurinol ---- Used in the treatment of Gout

Xanthine Oxidase Hypoxanthine Uric Acid Allopurinol

Sulphonamides:

Used as antibacterial agents.Similar in structure to PABA

For Folate synthesis PABA is essential Sulphonamide Needed for Bacterial Growth

Methotrexate

Methotrexate is 4-amino N10 methyl folic acid.

Used in cancer therapyMethotrexate resembles folic acid it

competitively inhibits “Folate Reductase”Prevents the formation of FH4

DNA Synthesis is inhibited

MAO Inhibitors MAO inhibitors are Ephedrine and

Amphetamine

Enzyme Mono Amine Oxidase oxidizes Epinephrine and Nor-epinephrine

MAO inhibitors competitively inhibit MAO ,prolong action of presser amines.

PhysotigminePhysotigmine is Acetylcholine esterase

inhibitorAcetylcholine Acetate + Choline

This drug prevents destruction of Acetylcholine,

Continued presence of Acetylcholine in post synaptic regions prolong neural impulse.

Dicoumarol

• Dicoumarol is used as an anticoagulant

• It competitively inhibits Vitamin K

Clinically useful Competitive Inhibitors

Drug Enzyme Inhibited Clinical use

Penicillin Transpeptidase Bacteria

Sulphonamide Pteroid synthetase Bacteria

6-Mercapto Purine Adenylo succinate Synthetase

Cancer

Neostigmine Acetyl Ch.esterase MyastheniaAlpha methyl DOPA DOPA Decarboxylase Hypertension

Lovastatin HMG Co A-reductase

Cholesterol Lowering

Non-Competitive InhibitionNo competition between the Inhibitor and

substrate.These inhibitors do not resemble the

substrate and bind to a site away from the active site.

Enzyme inhibitor has normal affinity for the substrate but produce products at a decreased rate.

Non-Competitive Inhibition

In Non-Competitive Inhibition (i) Affinity Remains the same

(ii) Efficiency decreases

(iii) 1/ Km remains the same as substrate concentration has no effect on the inhibitor

(iv) 1/ Vmax Increases as V has a decrease

Lineweaver Burk’s Plot for Non-Competitive Inhibition

1/[s]1/ Km

1 / v

1 / Vmax

1 / Vmax

+ Inhibitor

No Inhibitor

Non-competitive inhibition can be reversed if the inhibitor can be removed without affecting the enzyme activity.

Eg: Enzymes with –SH groups bind to heavy metals like Hg , Pb, resulting in non-competitive inhibition.

It can be reversed not by high levels of substrate but by increasing –SH in the medium.

Uncompetitive InhibitionIn this case the Inhibitor does

not bind to either enzyme or substrate, but combines with the ES complex to form ESI complex.

E + S ES + I ESI (no product forms)

Degree of inhibition increases with increase in substrate concentration.

There is a change in Vmax .It is lowered

This type of inhibition is very rare.

Uncompetitive Inhibition+

1/V

1/V

1/V

1/ [s]

1/V

1/Km 1/Km 1/Km

More Inhibitor

+ Inhibitor

Uncompetitive Inhibition

Suicide InhibitionFrom all the discussions it can

be assumed that the inhibitor binds to the enzyme reversibly.

The inhibitor just sits there, either in the active site or in a second binding site, but it is free to go back to solution.

There are another type of inhibitors that undergo irreversible chemistry with certain key amino acid residues (or prosthetic groups) in the active site, therefore killing the enzyme's activity, and preventing the inhibitor from being released out of the active site.

Many of these molecules are very effective drugs, because they are targeted specifically for a certain enzyme and kill the enzymes for good.

This inhibitors kill the enzyme for

good, but since they also 'die' in the process, they are called suicide or mechanism-based inhibitors.

Suicide inhibition It is a type of irreversible inhibition The inhibitor makes use of an enzyme’s

own reaction mechanism to inactivate itIn suicide inhibition, the structural

analogue is converted to a more effective inhibitor with the help of the enzyme to be inhibited.

This new product binds to the enzyme and inhibits further reaction.

Eg : Of Suicide inhibition1.Allopurinol a competitive inhibitor

for enzyme xanthine oxidase

When it comes in contact with the enzyme it is oxidized by xanthine oxidase to alloxanthine which is a stronger irreversible inhibitor of the enzyme

2. Anti-inflammatory Action of Aspirin

Membrane bound phospholipids are broken down first to Arichidonic acid (by phospholipases)

CyclooxygenaseArichidonic Acid Prostaglandins

Aspirin acetylates a serine residue in the active center of cyclo-oxygenase ,inhibiting prostaglandin synthesis and reducing inflammation

3. Difluromethyl ornithine against sleeping sickness TrypanosomiasisOrnithine Decarboxylase converts

Ornithine to putriscine a polyamineWhen this enzyme ODC

inTrypanosoma(parasite) is inhibited, multiplication of the parasite is arrested.

DFMO is initially inert. Binding with the enzyme it forms an irreversible covalent complex with co-enzyme PyPO4 and amino acid residues

S.No ENZYME Allosteric Inhibitor

Allosteric activator

1 HMG Co A -reductase Cholesterol

2 Phosphofructokinase ATP,Citrate AMP,F2,6,P

3 Pyruvate Carboxylase ADP Acetyl Co A4 Acetyl CoA Carboxylase AcylCoA Cirate

5 Citrate Synthase ATP

6 Carbamyl Phosphate Synthetase- I N-Acetyl Glut

7 Carbamyl PhosphateSynthetase-II UTP

8 Aspartate Transcarbamylase CTP ATP