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

CHMI 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

CHMI 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;

CHMI 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

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

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

CHMI 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

CHMI 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…);

CHMI 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;

CHMI 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

CHMI 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;

CHMI 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

CHMI 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

CHMI 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

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

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

CHMI 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.