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Enzyme regulation by Allosteric control (include feedback inhibition)

Enzyme Regulation - Regulatory Strategies. Enzyme regulation by Allosteric control (include feedback inhibition) Stimulation and inhibition by control proteins Reversible covalent modification Proteolytic activation. Allosteric Control Example: Feedback Inhibition. Feedback inhibition

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Enzyme regulation by Allosteric control (include feedback inhibition)

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  1. Enzyme Regulation - Regulatory Strategies • Enzyme regulation by • Allosteric control (include feedback inhibition) • Stimulation and inhibition by control proteins • Reversible covalent modification • Proteolytic activation

  2. Allosteric ControlExample: Feedback Inhibition • Feedback inhibition • Seen in multi-enzyme systems • One enzyme acts as a regulatory enzyme • When end product exceeds cell’s requirement, it inhibits specifically the regulatory enzyme • All other enzymes in the system are slowed as a result of lowered substrate level. • Example: E1 of the L-Ile biosynthesis (from L-Thr) enzyme system • E1: Threonine dehydratase, is specifically inhibitedallosterically by L-Ile, the end of product, but not by any of the four intermediates • L-Ile binds at a regulatory not the active site .

  3. Activation of Protein Kinase A by cAMP (Allosteric Control) • Protein Kinase A (PKA) • Alters activities of target proteins by phosphorylating specific Ser/Thr • Activated by cAMP • Mechanism of activation (allosteric) • R2C2 (in the absence of cAMP) • 2 catalytic unit, high affinity • 2 regulatory unit, high affinity for cAMP • R2 and 2C (in the presence of cAMP) • R chain binds C chain • pseudosubstrate sequence (Arg-Arg-Gly-Ala-Ile) in R • Blocks active site of C • cAMP binding • Induces conformational change in R • Causes dissociation of C from R (unblock active site)

  4. Covalent Modification • Some regulatory enzymes undergo reversible covalent modification • Modifying groups • Phosphoryl (-PO32-) (on Tyr, Ser, Thr, His) • adenylyl (Tyr) • uridylyl (Tyr) • adenosine diphosphate ribosyl (Arg, Gln, Cys, diphthamide - a modified His) • methyl groups (Glu) • Covalently linked to and removed from the regulatory enzyme by separate enzymes

  5. Enzyme Regulation by Phosphorylation • Phosphorylation carried out by protein kinases R-OH + ATP R-O-PO32- + ADP + H+ • Protein dephosphorylation carried out by protein phosphatases R-O-PO32- + H2O R-OH + Pi • Phosphorylation • Most common type of reversible covalent modification • Affects structure and therefore regulates activities of many enzymes Residues that can be phosphorylated: Ser, Thr, Tyr

  6. Effects of Phosphylation(Phosphoryl groups affect the structure and catalytic activity of proteins) • Adds to negative charges  electrostatic interaction  structural changes  change in substrate binding or catalytic activity (e.g., repel negative charges on Glu/Asp or favorable interaction either electrostatic or H-bonding with Arg) • Phosphoryl group capable of 3 H-bonds, highly directional • Phosphorylation is fast  enzyme can be turned on/off fast • Example: glycogen phosphorylase

  7. Glycogen Phosphorylase • Glycogen Phosphorylase aandbdiffer in their secondary, tertiary, and quaternary structures • The active site undergoes changes in structure and, consequently changes in catalytic activity as a consequence of phosphorylation /dephosphorylation • How? N-term 20 amino acids (contains basic residues such as Arg) interact withacidic residues somewhere else. Phosphorylation of Ser14disrupts these interaction and results in conformational change. Less active Ser Ser More active

  8. Ser phosphorylation site (Yellow) Allosteric activator AMP (dark blue) Subunit 2 Phosphorylase a Active site Subunit 1 Pyridoxal phosphate (PLP, light blue) (Vit B6 derivative) Glucose (red) bound at active site

  9. Proteolytic Activation: Zymogen to Active Protease • Activation of proteases • Pepsinogen (stomach) to pepsin • Trypsinogen (pancrease) to trypsin • Chymotrypsinogen (pancrease) to chymotrypsin • Procarboxypeptidase (pancrease) to carboxypeptidase • Proelastase (pancrease ) to elastase • Blood clotting enzymes • Proinsulin(protein hormone) to insulin • Procollagenase to collagenase • Activation of zymogens in the control of developmental processes

  10. Trypsinogen is the Common Activator of All the Pancreatic Zymogens • Concurrent action of digestive proteases in duodenum • Trypsin activates: trypsinogen chymotrypsinoge proelastase procarboxypeptidase • What activates trypsinogen first to produce trypsin? • Enteropeptidase (secreted by duodenum)

  11. Chymotrypsinogen Activationslide 1 • Chymotrypsinogen (inactive)  chymotrypsin (active) • chymotrypsinogen (245aa single chain) to -chymotrypsin (active) by trypsin • -chymotrypsin to -chymotrypsin by chymotrypsin

  12. Mechanism of Chymotrypsinogen Activationslide 2 • Newly formed N-term (Ile16) (+ly charged) turns inward and interacts with Asp194 • Induces conformational change • Incomplete substrate binding site becomes complete • Summary: Hydrolysis of a single peptide bond results in highly localized conformational changes that switches the enzymatic activity of the enzyme

  13. Pancreatic Trypsin Inhibitor • Zymogen (inactive)  active enzyme • Proteolysis (irreversible) • Active enzyme  inactive enzyme • Protease inhibitors • Pancreatic trypsin inhibitor (6 kDa) • Kd = 0.1 pM (tight binding to trypsin) • Complex cannot be dissociated with 8 M urea or 6 M guianidine • A very effective substrate analog • Almost perfectly complementary to active site

  14. 1-antitrypsin and Pulmonary Emphysema • 1-antitrypsin (1-antiproteinase) (plasma protein) • Protects tissue from digestion by elastase (secreted by neutrophils, white blood cells that engulf bacteria) • Blocks elastase much better than trypsin • 1-antitrypsin deficiency and emphysema • Genetic disorders lead to 1-antitrypsin deficiency • Excess elastase digests elastic fibers (elastin & collagen type IV) and other connective tissue proteins • This destroys alveolar walls in the lungs  emphysema • Cigarette smoking increases the likelihood of develop emphysema • Smoke oxidizes Met358 of the inhibitor, essential for elastase binding

  15. Pepsinogen Activation • Pepsin (digest proteins in the highly acidic environment of the stomach) • pH optimum 2 • Catalytic residues: 2 Asp • First 44 amino acids removed proteolytically and spontaneously below pH 5 • Mechanism of activation • Highly basic precursorsegment (6 Arg&Lys, + charged) • Highly acidic pepsin moiety(including catalytic Asp residues) • Fully formed active site is blocked in zymogen form • Lowering pH below 5 disrupts salt bridges and exposes active site

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