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

2. 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;

3. CHMI 2227 - E.R. Gauthier, Ph.D.3 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! British Journal of Cancer (2005) 92(Suppl 1), S6 – S1 Endothelial cell VEGF VEGF-R Endothelial cell growth/migration ZD6474

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

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

6. CHMI 2227 - E.R. Gauthier, Ph.D.6 Trypsin inhibitor 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:  At high enough [S], S will outcompete I Km is increased (Km app ):  Because I can bind E, the amount of S required to reach ½ Vmax will be increased. P

7. CHMI 2227 - E.R. Gauthier, Ph.D.7 Reversible Enzyme inhibition 1- Competitive inhibition The value of Km app can be used to obtain Km and Ki (the dissociation constant for the inhibitor):  Km app = 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.

8. CHMI 2227 - E.R. Gauthier, Ph.D.8 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.

9. CHMI 2227 - E.R. Gauthier, Ph.D.9 Reversible Enzyme inhibition 2- Uncompetitive inhibition Vmax app = Vmax / (1 + [I]/Ki) Km app = Km / (1 + [I]/Ki)

10. CHMI 2227 - E.R. Gauthier, Ph.D.10 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).

11. CHMI 2227 - E.R. Gauthier, Ph.D.11 Reversible Enzyme inhibition 3- Noncompetitive inhibition Vmax app = Vmax / (1 + [I]/Ki)

12. 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

13. CHMI 2227 - E.R. Gauthier, Ph.D.13 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)

14. CHMI 2227 - E.R. Gauthier, Ph.D.14 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;

15. CHMI 2227 - E.R. Gauthier, Ph.D.15 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;

16. CHMI 2227 - E.R. Gauthier, Ph.D.16 Irreversible enzyme inhibition 3. 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; Structure of the bacterial cell wall Sugars Tetrapeptide pentaGly bridges Pen

17. CHMI 2227 - E.R. Gauthier, Ph.D.17 Irreversible enzyme inhibition 3. Suicide inhibitors - penicillin Tetrapeptide pentaGly bridge Glycopeptide transpeptidase Glycopeptide transpeptidase Glycopeptide transpeptidase Penicillin

18. CHMI 2227 - E.R. Gauthier, Ph.D.18 Irreversible enzyme inhibition 3. Suicide inhibitors - penicillin