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CHMI 2227E Biochemistry I Enzymes: - Regulation CHMI 2227 - E.R. Gauthier, Ph.D. 1 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: 1-Allostery Inhibition by product Activation by substrate/cofactor 2- Binding of regulatory subunits 3- Covalent modification: Phosphorylation on Ser/Thr/Tyr 4- Degradation of the enzyme 5- Limited proteolysis CHMI 2227 - E.R. Gauthier, Ph.D. 2 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; CHMI 2227 - E.R. Gauthier, Ph.D. 3 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 Carbomoyl phosphate Asp CHMI 2227 - E.R. Gauthier, Ph.D. 4 Regulation of enzyme activity 1. Allostery - ATCase CHMI 2227 - E.R. Gauthier, Ph.D. 5 Regulation of enzyme activity 1. Allostery - ATCase CTP ATCase CHMI 2227 - E.R. Gauthier, Ph.D. 6 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 Caffeine AMP CHMI 2227 - E.R. Gauthier, Ph.D. cAMP Phosphodiesterase Adenylate Cyclase 7 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…); CHMI 2227 - E.R. Gauthier, Ph.D. 8 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; CHMI 2227 - E.R. Gauthier, Ph.D. 9 Regulation of enzyme activity 3. Phosphorylation Adrenaline Adrenaline receptor Adenylate cyclase Adrenaline Outside the cell G Protein g b a g ↑Energy Run Like Hell! a Inside the cell a cAMP Glycogen (glucose stores) ↑Intracellular glucose b Glycogen Phosphorylase-PO4 Phosphorylase kinase ATP Protein Kinase A (inactive) PKA-cAMP (active) Phosphorylase Kinase-PO4 Glycogen CHMI 2227 - E.R. Gauthier, Ph.D. Phosphorylase 10 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; CHMI 2227 - E.R. Gauthier, Ph.D. 11 Regulation of enzyme activity 4. Regulation of enzyme stability Ub E1 76 amino acid protein Tags other proteins for degradation; Ub Enzyme E1 E2 Ub E2 E3 Ub E3 Ubiquitin: Ubiquitin is attached to other proteins by a series of 3 enzymes (E1, E2 and E3); Enzyme Ub Ub Ub Ub CHMI 2227 - E.R. Gauthier, Ph.D. 12 Regulation of enzyme activity 4. Regulation of enzyme stability Polyubiquitylated proteins are targeted to a HUGE protein complex called the proteasome: Ub Ub Ub Ub Ub Ub 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!); CHMI 2227 - E.R. Gauthier, Ph.D. Ub Ub Contains several subunits with protease (i.e. protein hydrolases) activities; 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 Proteasome Degraded enzyme 13 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 CHMI 2227 - E.R. Gauthier, Ph.D. 14 Regulation of enzyme activity 5. Limited proteolysis CHMI 2227 - E.R. Gauthier, Ph.D. 15 Regulation of enzyme activity 5. Limited proteolysis Small intestine Secreted by pancreas CHMI 2227 - E.R. Gauthier, Ph.D. 16 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 (a1-antitrypsin, which inhibits elastase), leads to damage to the lungs and emphysema. Cigarette smoke also oxidizes a very important side chain of a1antitrypsin, leading ot its inactivation and emphysema. CHMI 2227 - E.R. Gauthier, Ph.D. 17