Enzymology :Types of Enzyme inhibition & Therapeutic uses

Dr. Rohini C Sane

Enzyme inhibitionEnzyme inhibition

• An Inhibitor –a chemical agent inhibiting /poisoning enzyme

Types of Enzyme inhibition

1. Irreversible Enzyme inhibition

2. reversible Enzyme inhibition

Classification of enzyme inhibition

NO PRODUCT FORMATION

Comparison of competitive and non-competitive enzyme inhibition

Allosteric Regulation

Change of three dimensionalof enzyme by Allosteric inhibitor & activator

Irreversible inhibition• Irreversible inhibitor : destroys a functional group on enzyme

necessary for catalytic activity

• eg Di Isopropyl Fluorophosphate(DFP ) inhibits Acetyl choline esterase

• Iodoacetamide inhibits OH group of serine ,SH group of Cysteine ,Imidazole group of Histidine

DFP – inhibits Serine Proteases such as Acetyl Choline Esterase , Trypsin ,Chymotrypsin ,Elastase

BAL –BRITISH ANTI LEVISITE- Antidote for HEAVY METALS

CYANIDE –Cytochrome Oxidase

FLUORIDE –inhibits Enolase ( removes Mg ⁺⁺/Mn⁺⁺ from the active site OF enzymes

Irreversible inhibition of enzymes

Neurotransmission at synaptic junction by neurotransmitters

Irreversible inhibition of Acetyl Choline Esterase

Malathione – an insecticide

Inhibition of Neurotransmission at synaptic junction by inhibitors of neurotransmitters-Donepezil

Irreversible inhibition of enzymes with SH group -by Iodoacetamide

enzymes with SH group + Iodoacetamide

reversible inhibition of enzymes with Chemically modified enzyme ( inactive )

Allosteric Regulation a) Allosteric inhibitor b) Allosteric activator

Allosteric Regulation a) Allosteric inhibitor b) Allosteric activator

Allosteric Regulation a) Allosteric inhibitor-binds to anallosteric site on enzyme &induces conformation change preventingSubstrate to bind to an active site on enzyme .Product formation inhibited .

Allosteric Regulation a) Allosteric inhibitor-

three dimensional change in enzyme molecule &an active site of enzyme deformed.

Allosteric & covalent modulation

Activation of manyenzymes involve

Phosphorylation of enzyme molecule by Protein Kinases.Deactivation of many enzymes is facilitated by dephosphorylizationof enzyme by

Phosphoprotein Phosphatases

Reversible inhibition of enzymes by Competitive inhibitors

Competitive inhibitors

1. Competitive inhibitors competes with substrate for binding to active site but once bound substrate cannot be transformed into product by enzymes.

2. Inhibition by Competitive inhibitors can be reversed by simply increasing concentration of substrate

3. Competitive inhibitors resembles the normal substrate in 3D structure

4. E + I = EI

NO PRODUCT FORMATION

Comparison of competitive and non-competitive inhibition Competitiveinhibitorsresembles the normal substrate in 3D structure &non-Competitiveinhibitors don’t resembles the normal substrate in 3D structure

COMPETITIVE INHIBITION ---Competitive inhibitors1 .competes with substrate for binding to active site but once bound substrate cannot be transformed into product by enzymes. 2.Inhibition by Competitive inhibitorscan be reversed by simply increasing concentration of substrate

Changes in Reaction Rate in presence of Competitive & Non –competitive inhibitor

Inhibition by Competitive inhibitorscan be reversed by simply increasingconcentration of substrate& that by

non -Competitive inhibitorscann’t be reversed by simply increasing substrate concentration.

Competitive inhibition

Competitive inhibitors competes with substrate for binding to active site but once bound substrate cannot be transformed by enzymes into product.

COMPETITIVE INHIBITION OF SUCCINATE DEHYDROGENASE BY FUMARATE ,MALONATE ,OXALO ACETATE

Structure of Succinic acid ,Malonic acid & oxaloacetic acid with two carboxylic acid groups (COOH )& can bind to an active site on succinate dehydrogenase –Competitive inhibitors

Clinical significance of Competitive inhibition

Clinical significance of Non competitive inhibitors

LineWeaver equation & enzyme inhibition

THERAPEUTIC USES OF COMPETITIVE INHIBITORS-Anti Cancer Therapy /Drugs for MyostheniaGravis/Antihypertensive

Non competitive Inhibition

Characteristics of Non competitive Inhibition

1. Reversible but not reversed by substrate

2. Inhibitor binds at site other than substrate binding site

3. It binds reversibly to both free enzyme & ES complex to form inactive complex EI & ESI

E + I ↔ EI

ES + I ↔ ESI

4. Inhibitors alter the conformation of E molecule so that reversible activation occurs

5. They are naturally occurring metabolic intermediates .

Non competitive inhibition

Threonine lsoleucine ( Threonine Dehydratase inhibited by isoleucine-final product of cascade /pathway ) -feed back inhibition

Comparison between competitive & Non competitive inhibition

Criteria competitive Non competitive

Active on Active site May or may not be active at binding site

Structure of inhibitors Substrate analogs Unrelated molecules

Inhibition reversible Generally irreversible

Excess of substrate Inhibition relieved No effect

Km Increased in presence of inhibitor

Unchanged in presence of inhibitor

Vmax Unchanged decreased

Significance Therapeutic application Toxicological application

Comparison between competitive & Non competitive inhibition

Competition inhibition –Vmax unchanged & Km INCREASEDNon competitive inhibition –Vmax decreased & Km not altered

Comparison between competitive & non competitive inhibition

Catalytic site

Regulatory site

Isoleucine Binds toregulatory site onThreonine Dehydratase & functions as non-competitive inhibitor

Mechanism of action of Aspirin

Uncompetitive inhibition of enzymes

Uncompetitive inhibition- inhibitors binds to ES ComplexVmax & Km decreased eg Alkaline Phosphatase by phenylalanine

Suicide InhibitionProperties of Suicide Inhibition

1. Irreversible inhibition

2. More effective inhibitor

3. synthesized with the help of enzyme

4. Inhibition of xanthine oxidase by Allopurinol (treatment of GOUT )

5. Alloxanthine synthesized by xanthine oxidase using Allopurinol is more potent inhibitor of enzyme than Allopurinol

6. Arachidonic acid Prostaglandin ( cyclo oxygenase )is inhibited by Aspirin.(anti inflammatory )

7. 5 Fluoro-Uracil 5 Fluoro Deoxy Uridylate more potent inhibitor of Thymidylate synthase to inhibit nucleotide synthesis and is being used in cancer treatment

Properties of Suicide inhibition

Suicide Inhibition of Allopurinol

Mechanism of action of 5-Fluorouracil

Competitive inhibitors-Therapeutic usesInhibitor (drug ) Enzyme inhibited Disease treated

Allopurinol Xanthine Oxidase Gout

Epidrene (MAO INHIBITOR )

Mono Amino Oxidase Psychiatric Treatment

Succinyl CoA Acetyl CoA Anesthesia

Dicumarol VITAMIN K EPIOXIDE REDUCTASE ANTICOAGULANT

Lovastatin HMG CoA reductase Reducing Cholesterol levels

INH ( Isonicotinic acid hydrazide )

Pyridoxal phosphate Tuberculosis

Neostigmine Acetyl Choline Esterase Myasthenia Gravis

Alpha Methyl Dopa Dopa Carboxylase Myasthenia Gravis

Competitive inhibitors-Therapeutic usesInhibitor (drug ) Enzyme inhibited Disease treated

Penicillin Trans peptidase bactericidal

Sulphonamide ( analog PABA ) Steroid synthatase Bactericidal

Trimethoprim FH2 reductase bactericidal

Pyri methamine FH2 reductase bactericidal

Methotrexate FH2 reductase Leukemia

6 –Mercapto purine Adenylo Succinate Synthtase Cancer

5 –Fluoro Uracil Thymidylate Synthtase Cancer

Azo Serine Phospho Ribosyl Amido Transferase Cancer

Cytosine Arabinoside DNA Polymerase Cancer

ACYCLOVIR DNA Polymerase Cancer

Therapeutic usesOf enzyme inhibitors