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CHMI 2227E Biochemistry I Enzymes: - Inhibition CHMI 2227 - E.R. Gauthier, Ph.D. 1 Enzyme inhibition Enzyme inhibitors inactivate the enzyme; Two main types of inhibition exist: Reversible enzyme inhibition: enzyme activity can be recovered by removing the inhibitor (e.g. dialysis, gel filtration); Irreversible enzyme inhibition: inhibitor binds covalently to enzyme, which is then irreversibly inactivated. The inhibition (i.e. inactivation) of an enzyme can tell us a lot about the way it works; Enzyme inhibitors are frequently used to define biological phenomena; Enzyme inhibitors are also sought by the big pharma to block enzymes involved in diseases; CHMI 2227 - E.R. Gauthier, Ph.D. 2 Enzyme inhibition Example 1 - VEGF Receptor inhibitors: VEGF (Vascular Endothelial Growth Factor): Produced in embryos and tumours; Acts via a cell surface receptor to trigger the growth of blood vessels; Why inhibit VEGF-R: Blocking the action of VEGF (an enzyme) will block the growth of blood vessels and starve tumours to death! VEGF-R VEGF Endothelial cell growth/migration ZD6474 Endothelial cell CHMI 2227 - E.R. Gauthier, Ph.D. of Cancer (2005) 92(Suppl 1), S6 – S1 British Journal 3 Int. J. Impot. Res. (2004) 16, S11–S14 Enzyme inhibition Example 2 – Sildenafil: Sildenafil: cGMP-Phosphodiesterase inhibitor; Initially tested as an antihypertension drug; Acetylcholine Endothelial cell Vascular smooth muscle cell GMP PDE Nitric Oxide Synthase (NOS) Arginine Muscle relaxation Sildenafil cGMP NO NO Guanylate cyclase CHMI 2227 - E.R. Gauthier, Ph.D. Blood vessel Dilation GTP 4 Enzyme inhibition Example 3 – Acetaminophen (tylenol): CHMI 2227 - E.R. Gauthier, Ph.D. 5 Reversible Enzyme inhibition 1- Competitive inhibition Most frequently encountered inhibitors; I is very similar to S (i.e. it is a structural analog) I and S compete for the same binding site on the enzyme: the active site; Vmax stays the same: P At high enough [S], S will outcompete I Km is increased (Kmapp): Because I can bind E, the amount of S required to reach ½ Vmax will be increased. Trypsin inhibitor CHMI 2227 - E.R. Gauthier, Ph.D. 6 Reversible Enzyme inhibition 1- Competitive inhibition The value of Kmapp can be used to obtain Km and Ki (the dissociation constant for the inhibitor): Kmapp = Km (1 + [I]/Ki) Ki = [E][I]/[EI] Ki is a measure of the affinity of I for E: the smaller Ki, the more potent the inhibition. CHMI 2227 - E.R. Gauthier, Ph.D. 7 Reversible Enzyme inhibition 2- Uncompetitive inhibition I only bind to ES, not the free enzyme; Example: glycophosphate (Round-up herbicide) Vmax is decreased: Some of the E is converted into an inactive ESI complex. Km is decreased: I reduces the amount of E that can participate in the reaction; ESI shifts the E + S ES to the right, leading to an apparent decrease in Km. CHMI 2227 - E.R. Gauthier, Ph.D. 8 Reversible Enzyme inhibition 2- Uncompetitive inhibition Vmaxapp= Vmax / (1 + [I]/Ki) Kmapp= Km / (1 + [I]/Ki) CHMI 2227 - E.R. Gauthier, Ph.D. 9 Reversible Enzyme inhibition 3- Noncompetitive inhibition I and S bind to different sites on E; Binding of I on E doesn’t affect the binding of S on E (and vice versa); So: Km is unchanged, but Vmax is decreased (I reduces the [E] that can generate P); E.g. deoxycyclin (an antibiotic), which inhibits collagenase (a proteolytic enzyme involved in periodontal diseases). CHMI 2227 - E.R. Gauthier, Ph.D. 10 Reversible Enzyme inhibition 3- Noncompetitive inhibition Vmaxapp= Vmax / (1 + [I]/Ki) CHMI 2227 - E.R. Gauthier, Ph.D. 11 Irreversible enzyme inhibition Irreversible inhibitors bind covalently to the enzyme and permanently inhibit it. Very useful to identify the amino acids involved in catalysis Three types: Group-specific Active site-directed reagents (aka Affinity labels) Suicide inhibitors CHMI 2227 - E.R. Gauthier, Ph.D. 12 Irreversible enzyme inhibition 1. Group-specific inhibitors React with amino acid side chains; Lead to inhibition by interfering with the catalysis (e.g. by reacting with side-chains important for the catalysis); E.g. diisopropyl fluorophosphate (DFP); Nerve gas Inhibits acetylcholine esterase (and many other proteases with Ser at the active site) CHMI 2227 - E.R. Gauthier, Ph.D. 13 Irreversible enzyme inhibition 2. Affinity labels Inhibitor is structurally similar to S; Reacts with active site residues; I reacts with E to form a covalent bond that cannot be hydrolysed; CHMI 2227 - E.R. Gauthier, Ph.D. 14 Irreversible enzyme inhibition 3. Suicide inhibitors Modified substrates; Initially processed by E as if it were the normal S; However, an reaction intermediate covalently and irreversibly binds the E, leading to its inhibition; Example 1: monoamine oxidase (MAO) inhibitors (MAO – breaks down certain neurotransmitters, e.g. serotonine, adrenaline) high MAO activity = depression; CHMI 2227 - E.R. Gauthier, Ph.D. 15 Irreversible enzyme inhibition 3. Suicide inhibitors - penicillin Tetrapeptide Sugars Interfere with the synthesis of the bacterial cell wall Makes bacteria much less resistant to stress; pentaGly bridges Pen Cell wall: Peptidoglycan Penicillin blocks the formation of the link between the tetrapeptide and the pentaGly bridge; Structure of the bacterial cell wall CHMI 2227 - E.R. Gauthier, Ph.D. 16 Irreversible enzyme inhibition 3. Suicide inhibitors - penicillin Glycopeptide transpeptidase Glycopeptide transpeptidase pentaGly bridge Penicillin Tetrapeptide Glycopeptide transpeptidase CHMI 2227 - E.R. Gauthier, Ph.D. 17 Irreversible enzyme inhibition 3. Suicide inhibitors - penicillin CHMI 2227 - E.R. Gauthier, Ph.D. 18