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CHMI 2227E Biochemistry I. Enzymes: Regulation. 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;
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CHMI 2227EBiochemistry I Enzymes: Regulation CHMI 2227 - E.R. Gauthier, Ph.D.
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 Regulation of enzyme activity • 1-Allostery • Inhibition by product • Activation by substrate/cofactor • 2- Binding of regulatory subunits CHMI 2227 - E.R. Gauthier, Ph.D.
Regulation of enzyme activity1. 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.
ATCase Carbomoyl phosphate Asp Regulation of enzyme activity1. 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); CHMI 2227 - E.R. Gauthier, Ph.D.
Regulation of enzyme activity1. Allostery - ATCase CHMI 2227 - E.R. Gauthier, Ph.D.
CTP ATCase Regulation of enzyme activity1. Allostery - ATCase CHMI 2227 - E.R. Gauthier, Ph.D.
ATP Adenylate Cyclase cAMP cAMP Phosphodiesterase Caffeine AMP Regulation of enzyme activity2. 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; CHMI 2227 - E.R. Gauthier, Ph.D.
Regulation of enzyme activity3. 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.
Regulation of enzyme activity3. 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.
Adrenaline receptor Adenylate cyclase Adrenaline Outside the cell Inside the cell G Protein ATP cAMP Glycogen (glucose stores) Protein Kinase A (inactive) Phosphorylase kinase Glycogen Phosphorylase-PO4 a a a g g b b ↑Intracellular glucose PKA-cAMP (active) ↑Energy Phosphorylase Kinase-PO4 Glycogen Phosphorylase Run Like Hell! Regulation of enzyme activity3. Phosphorylation Adrenaline CHMI 2227 - E.R. Gauthier, Ph.D.
Regulation of enzyme activity4. 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.
Ub E1 Ub Enzyme E1 E2 Ub E2 E3 E3 Ub Enzyme Ub Ub Ub Ub Regulation of enzyme activity4. 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); CHMI 2227 - E.R. Gauthier, Ph.D.
Enzyme Ub Ub Ub Ub Ub Ub Ub Ub Proteasome Degraded enzyme Regulation of enzyme activity4. 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. CHMI 2227 - E.R. Gauthier, Ph.D.
Regulation of enzyme activity5. 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.
Regulation of enzyme activity5. Limited proteolysis CHMI 2227 - E.R. Gauthier, Ph.D.
Small intestine Secreted by pancreas Regulation of enzyme activity5. Limited proteolysis CHMI 2227 - E.R. Gauthier, Ph.D.
Regulation of enzyme activity5. 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 a1-antitrypsin, leading ot its inactivation and emphysema. CHMI 2227 - E.R. Gauthier, Ph.D.
