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

2. CHMI 2227 - E.R. Gauthier, Ph.D.2 Regulation of enzyme activity In any organism, enzymes are always kept in check so that their activity can match the needs of the cell:  Enzymes can be activated: the reaction is stimulated to generate more product;  Enzymes can be inactivated (or inhibited): the reaction is slowed down to decrease the amount of product; Several strategies are used to modulate (i.e. activate or inhibit) enzymes:  3- Covalent modification: Phosphorylation on Ser/Thr/Tyr  4- Degradation of the enzyme  5- Limited proteolysis  1-Allostery Inhibition by product Activation by substrate/cofactor  2- Binding of regulatory subunits

3. CHMI 2227 - E.R. Gauthier, Ph.D.3 Regulation of enzyme activity 1. Allostery Widely used in metabolic enzymes:  Inhibition by the end product of a pathway;  Activation by a product generated early on in the pathway; Based on the principle of cooperativity:  The binding of a small molecule to the enzyme modifies the 3-D structure of the protein and alters its ability to catalyse the reaction;

4. CHMI 2227 - E.R. Gauthier, Ph.D.4 Regulation of enzyme activity 1. Allostery Example: Aspartate transcarbomoylase (ATCase):  Involved in the first of a series of reactions leading to the production of CTP; CTP (the end product) inhibits ATCase by allostery; ATP activates ATCase, also by allostery (competes with CTP for binding regulatory sites on ATCase); ATCase Asp Carbomoyl phosphate

5. CHMI 2227 - E.R. Gauthier, Ph.D.5 Regulation of enzyme activity 1. Allostery - ATCase

6. CHMI 2227 - E.R. Gauthier, Ph.D.6 Regulation of enzyme activity 1. Allostery - ATCase ATCase CTP

7. CHMI 2227 - E.R. Gauthier, Ph.D.7 Regulation of enzyme activity 2. Regulatory subunits cAMP is produced from ATP by the action of adenylate cyclase; The binding of cAMP to the regulatory subunit of PKA frees the catalytic subunits, which are now fully active; ATP cAMP Adenylate Cyclase cAMP Phosphodiesterase AMP Caffeine

8. CHMI 2227 - E.R. Gauthier, Ph.D.8 Regulation of enzyme activity 3. Regulation by covalent modification Specific amino acid side chains of several enzymes are the target of covalent modifications (catalysed by yet other enzymes…);

9. CHMI 2227 - E.R. Gauthier, Ph.D.9 Regulation of enzyme activity 3. Regulation by phosphorylation The addition of a phosphate group (phosphorylation) by protein kinases and their removal (by protein phosphatases) is frequently used to modulate the activity of enzymes;

10. CHMI 2227 - E.R. Gauthier, Ph.D.10 Regulation of enzyme activity 3. Phosphorylation  Adrenaline Adrenaline       G Protein Adrenaline receptorAdenylate cyclase  ATP cAMP Protein Kinase A (inactive) ↑Intracellular glucose PKA-cAMP (active) ↑Energy Run Like Hell! Glycogen (glucose stores) Phosphorylase kinase Phosphorylase Kinase-PO 4 Glycogen Phosphorylase-PO 4 Glycogen Phosphorylase Inside the cell Outside the cell

11. CHMI 2227 - E.R. Gauthier, Ph.D.11 Regulation of enzyme activity 4. Regulation of enzyme stability Proteins are constantly being made (i.e. synthesized) and destroyed (i.e. degraded); The tight regulation of protein synthesis and degradation participates to the regulation of enzyme activity;

12. CHMI 2227 - E.R. Gauthier, Ph.D.12 Regulation of enzyme activity 4. Regulation of enzyme stability Ubiquitin:  76 amino acid protein  Tags other proteins for degradation; Ubiquitin is attached to other proteins by a series of 3 enzymes (E1, E2 and E3); Enzyme Ub Enzyme Ub E1 E2 E3 E1 E2 Ub E3 Ub

13. CHMI 2227 - E.R. Gauthier, Ph.D.13 Regulation of enzyme activity 4. Regulation of enzyme stability Polyubiquitylated proteins are targeted to a HUGE protein complex called the proteasome:  Contains several subunits with protease (i.e. protein hydrolases) activities; The degradation of the enzyme will result in a decrease in the amount of the product of the reaction catalysed by the enzyme (well, duh!); This is an extremely important phenomenon: the cell cycle (cell growth  DNA synthesis  mitosis) is tightly regulated by the timely degradation of a series of proteins called cyclins. Enzyme Ub Degraded enzyme Proteasome

14. CHMI 2227 - E.R. Gauthier, Ph.D.14 Regulation of enzyme activity 5. Limited proteolysis Several enzymes (particularly digestive enzymes) are initially synthesized as inactive precursors (zymogens / proenzymes); Activation of the enzymes is done by the cleavage of a limited number of peptide bonds (usually 2-3); The mature enzyme is therefore made up of 2 to 3 chains, held together by disulfide bonds

15. CHMI 2227 - E.R. Gauthier, Ph.D.15 Regulation of enzyme activity 5. Limited proteolysis

16. CHMI 2227 - E.R. Gauthier, Ph.D.16 Regulation of enzyme activity 5. Limited proteolysis Small intestine Secreted by pancreas

17. CHMI 2227 - E.R. Gauthier, Ph.D.17 Regulation of enzyme activity 5. Limited proteolysis Pancreatic trypsin inhibitor inhibits trypsin, and prevents trace amounts of activated trypsin from triggered the proteolytic cascade in the pancreas/pancreatic ducts; Inherited deficiencies in a similar protease inhibitor (  -antitrypsin, which inhibits elastase), leads to damage to the lungs and emphysema. Cigarette smoke also oxidizes a very important side chain of  - antitrypsin, leading ot its inactivation and emphysema.