CHMI 2227E Biochemistry I

1. CHMI 2227EBiochemistry I Enzymes: Inhibition CHMI 2227 - E.R. Gauthier, Ph.D.

2. 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.

3. 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 Enzyme inhibitionExample 1 - VEGF Receptor inhibitors: CHMI 2227 - E.R. Gauthier, Ph.D. British Journal of Cancer (2005) 92(Suppl 1), S6 – S1

4. Sildenafil: cGMP-Phosphodiesterase inhibitor; Initially tested as an anti-hypertension drug; Vascular smooth muscle cell Endothelial cell Acetylcholine GMP PDE Muscle relaxation Arginine Blood vessel Dilation Sildenafil Nitric Oxide Synthase (NOS) cGMP GTP Guanylate cyclase NO NO Enzyme inhibitionExample 2 – Sildenafil: Int. J. Impot. Res. (2004) 16, S11–S14 CHMI 2227 - E.R. Gauthier, Ph.D.

5. Enzyme inhibitionExample 3 – Acetaminophen (tylenol): CHMI 2227 - E.R. Gauthier, Ph.D.

6. P Trypsin inhibitor Reversible Enzyme inhibition1- 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: • 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. CHMI 2227 - E.R. Gauthier, Ph.D.

7. Reversible Enzyme inhibition1- 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.

8. Reversible Enzyme inhibition2- 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.

9. Reversible Enzyme inhibition2- Uncompetitive inhibition • Vmaxapp= Vmax / (1 + [I]/Ki) • Kmapp= Km / (1 + [I]/Ki) CHMI 2227 - E.R. Gauthier, Ph.D.

10. Reversible Enzyme inhibition3- 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.

11. Reversible Enzyme inhibition3- Noncompetitive inhibition • Vmaxapp= Vmax / (1 + [I]/Ki) CHMI 2227 - E.R. Gauthier, Ph.D.

12. 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.

13. 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) Irreversible enzyme inhibition1. Group-specific inhibitors CHMI 2227 - E.R. Gauthier, Ph.D.

14. Irreversible enzyme inhibition2. 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.

15. 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; Irreversible enzyme inhibition3. Suicide inhibitors CHMI 2227 - E.R. Gauthier, Ph.D.

16. Sugars Tetrapeptide pentaGly bridges Pen Structure of the bacterial cell wall Irreversible enzyme inhibition3. Suicide inhibitors - penicillin • Interfere with the synthesis of the bacterial cell wall • Makes bacteria much less resistant to stress; • Cell wall: • Peptidoglycan • Penicillin blocks the formation of the link between the tetrapeptide and the pentaGly bridge; CHMI 2227 - E.R. Gauthier, Ph.D.

17. Glycopeptide transpeptidase Glycopeptide transpeptidase pentaGly bridge Penicillin Tetrapeptide Glycopeptide transpeptidase Irreversible enzyme inhibition3. Suicide inhibitors - penicillin CHMI 2227 - E.R. Gauthier, Ph.D.

18. Irreversible enzyme inhibition3. Suicide inhibitors - penicillin CHMI 2227 - E.R. Gauthier, Ph.D.