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Introductions

Introductions. Name Email address Favorite type of food Least favorite food Magazine you read most often All-time favorite movie Concert/performance you’d most want to see Favorite sport to play / favorite sport to watch People would be surprised to learn this about me.

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Introductions

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  1. Introductions • Name • Email address • Favorite type of food • Least favorite food • Magazine you read most often • All-time favorite movie • Concert/performance you’d most want to see • Favorite sport to play / favorite sport to watch • People would be surprised to learn this about me.

  2. Regulation of Metabolism NUTR 543 Advanced Nutritional Biochemistry Dr. David L. Gee

  3. Characteristics of Regulatory Enzymes • Catalyze a rate-limiting step • Catalyze a committed step • Early step unique to a pathway • Irreversible step • Requires energy • Often results in a phosphorylated compound

  4. Types of Regulatory Mechanisms • Non-covalent interactions • Covalent modifications • Changes in abundance of the enzyme

  5. Types of Regulatory Mechanisms • Non-covalent interactions • Covalent modifications • Changes in abundance of the enzyme

  6. Non-covalent InteractionsSubstrate availability • Non-regulatory enzymes generally exhibit hyperbolic kinetics (Michaelis-Menton) • At low substrate concentration, reaction rate proportional to substrate concentration • Regulatory enzymes generally exhibit sigmoidal kinetics (positive cooperativity) • Changes of substrate concentrations at normal physiological levels greatly alter reaction rate

  7. Non-covalent InteractionsAllosteric Regulation • Binding of allosteric effectors at allosteric sites affect catalytic efficiency of the enzyme

  8. Non-covalent InteractionsAllosteric Regulation • Allosteric Activators • Decrease Km (increases the enzyme binding affinity) • Increases Vmax (increases the enzyme catalytic efficiency)

  9. Non-covalent InteractionsAllosteric Regulation • Allosteric Inhibitors • Increases Km (decreases enzyme binding affinity) • Decreases Vmax (decreases enzyme catalytic efficiency)

  10. Molecues that act as allosteric effectors • End products of pathways • Feedback inhibition • Substrates of pathways • Feed-forward activators • Indicators of Energy Status • ATP/ADP/AMP • NAD/NADH • Citrate & acetyl CoA

  11. Non-covalent InteractionsProtein-Protein Interactions • Calmodulin (CALcium MODULted proteIN) • Binding of Ca++ to calmodulin changes its shape and allows binding and activation of certain enzymes

  12. Binding of calcium to Calmodulin changes the shape of the protein Unbound Calmodulin on left Calcium bound Calmodulin on right. Stars indicate exposed non-polar ‘grooves’ that non-covalently binds proteins

  13. Calmodulin • Extracellular [Ca] = 5 mM • Intracellular [Ca] = 10-4 mM • Most of Ca bound inside cells • Bound Ca can be released by hormonal action, nerve innervation, light, …. • Released Ca binds to Calmodulin which activates a large number of proteins

  14. Calmodulin plays a role in: • Muscle contraction • Inflammation • Apoptosis • Memory • Immune response…. • Metabolism • Activates phosphorylase kinase • Stimulates glycogen degradation during exercise

  15. Types of Regulatory Mechanisms • Non-covalent interactions • Covalent modifications • Changes in abundance of the enzyme

  16. Covalent Regulation of Enzyme ActivityPhosphorylation and Dephosphorylation • Addition or deletion of phosphate groups to particular serine, threonine, or tyrosine residues alter the enzymes activity

  17. Covalent Regulation of Enzyme ActivityLimited Proteolysis • Specific proteolysis can activate certain enzymes and proteins (zymogens) • Digestive enzymes • Blood clotting proteins • Peptide hormones (insulin)

  18. Covalent Regulation of Enzyme ActivityEnzyme Cascades • Enzymes activating enzymes allows for amplification of a small regulatory signal

  19. Types of Regulatory Mechanisms • Non-covalent interactions • Covalent modifications • Changes in abundance of the enzyme

  20. Changes in Enzyme Abundance • Inducible vs Constitutive Enzymes • Induction is caused by increases in rate of gene transcription. • Hormones activate transcriptional factors • Increase synthesis of specific mRNA • Increase synthesis of specific enzymes

  21. Hormones, Receptors, and Communication Between Cells • Hormones • chemical signals that coordinate metabolism • Hormone Receptors • Target tissues • Specific binding • Types • Intracellular receptors • Cell-surface receptors

  22. Hormones, Receptors, and Communication Between Cells • Intracellular receptors • lipid soluble hormones • Steroid hormones, vitamin D, retinoids, thyroxine • Bind to intracellular protein receptors • This binds to regulatory elements by a gene • Alters the rate of gene transcription • Induces or represses gene transcription

  23. Hormones, Receptors, and Communication Between CellsIntracellular Receptors

  24. Hormones, Receptors, and Communication Between Cells • Cell-surface receptors • Water soluble hormones • Peptide hormones (insulin), catecholamines, neurotransmitters • Three class of cell-surface receptors • Ligand-Gated Receptors • Catalytic Receptors • G Protein-linked Receptors

  25. Cell Surface ReceptorsLigand-Gated Receptors • Binding of a ligand (often a neurotransmitter) affects flow of ions in/out of cell • Gamma-amino butyric acid (GABA) binds and opens chloride channels in the brain • Valium (anti-anxiety drug) reduces the amount of GABA required to open the chloride channels

  26. Cell-Surface ReceptorsCatalytic Receptors • Binding of hormone activates tyrosine kinase on receptor which phosphorylates certain cellular proteins • Insulin receptor is a catalytic receptor with TYR Kinase activity

  27. Cell-Surface ReceptorsG Protein-Linked Receptors • Binding of hormone activates an enzyme via a G-protein communication link. • The enzymes produces intracellular messengers • Signal transduction • Second messengers activate protein kinases

  28. Intracellular Messengers:Signal Transduction Pathways • Cyclic AMP (cAMP) • Diacylglycerol (DAG) & Inositol Triphosphate (IP3) • Cyclic GMP (cGMP)

  29. G-Protein-Linked Receptors:The cAMP Signal Transduction Pathway • Two types of G-Proteins • Stimulating G protein (Gs) • Activate adenylate cyclase • Inhibitory G proteins (Gi) • Inhibit adenylate cyclase

  30. G Proteins • G proteins are trimers • Three protein units • Alpha • Beta • gamma

  31. Alpha proteins are different in Gs and Gi • Both have GTPase activity • Alpha proteins modify adenylate cyclase activity • AC stimulated by Alpha(s) when activated by a hormone • AC Inhibited by Alpha(I) when activated by other hormones

  32. Family of G Proteins • Binding of hormones to receptors causes: • GTP to displace GDP • Dissociation of alpha protein from beta and gamma subunits • activation of the alpha protein • Inhibition or activation of adenylate cyclase • GTPase gradually degrades GTP and inactivates the alpha protein effect (clock)

  33. The cAMP Signal Transduction Pathway • cAMP – intracellular messenger • Elevated cAMP can either activate or inhibit regulatory enzymes • cAMP activates glycogen degradation • cAMP inhibits glycogen synthesis • [cAMP] affected by rates of synthesis and degradation • Synthesis by adenylate cyclase • Degradation by phosphodiesterase • Stimulated by insulin • Inhibited by caffeine

  34. What does cAMP do?Activation of Protein Kinase A by cAMP • Protein kinase A • Activates or inhibits several enzymes of CHO and Lipid metabolism • Inactive form: regulatory+catalytic subunits associated • Active form: binding of cAMP disassociates subunits

  35. Clinical Case • 25 y.o. female vacationing in Costa Rica • Severe diarrhea, nearly comatose • Diarrhea resulting in fluid loss of ~800 ml/hr • Hypotensive (75/50) • Metabolic acidosis , low bicarbonate • Stool sample contained Vibrio cholerae • IV administration of fluids, tetracycline • Patient improves rapidly

  36. Cholera background information • Severe and rapid diarrheal disease • Caused by Vibrio cholerae • Commonly shock after 4-12 hrs after first symptoms, death 18 hrs – several days without rehydration therapy (subject can lose up to 20 liters of fluids) • Source is commonly contaminated water

  37. Choleramechanism of action • V. cholarae produces protein that attaches to intestinal epithelial cells • Delivery subunit B (blue) facilitates entry of subunit A into cell • Subunit A catalyzes ADP-ribosylation of the alpha-s subunit of Gs-protein

  38. Clinical Case • V. cholerae toxin affects alpha-S subunit • Inactivates GTP’ase • Alpha-S subunit permanently active • Stimulates adenylate cyclase • Overproduces cAMP • stimulates protein kinase • Phosphorylation of membrane ion transport proteins – massive losses of Na, Cl, K, HCO3

  39. Hypothetical link to cystic fibrosis • Cystic fibrosis characterized by • Salty sweat • Very thick mucous • Homozygous genetic defect to chloride transport to mucous • Decreased chloride results in less water following due to osmosis, leading to thicker mucous • Heterozygous mutation (normal mucous) has transport protein resistant to effects of cholera toxin ?

  40. Intracellular Messengers:Signal Transduction Pathways • Cyclic AMP (cAMP) • Diacylglycerol (DAG) & Inositol Triphosphate (IP3) • Cyclic GMP (cGMP)

  41. DAG & IP3Phosphotidylinositol Signal Transduction Pathway • Hormone activation of phospholipase C • Via Gp protein • Phospholipase C hydrolyzes membrane phospholipids (phosphotidyl inositol) to produce DAG and IP3

  42. DAG & IP3Phosphotidylinositol Signal Transduction Pathway • IP3 stimulates release of Ca from ER • Protein kinase C activated by DAG and calcium

  43. Intracellular Messengers:Signal Transduction Pathways • Cyclic AMP (cAMP) • Diacylglycerol (DAG) & Inositol Triphosphate (IP3) • Cyclic GMP (cGMP)

  44. cGMPThe cGMP Signal Transduction Pathway • cGMP effects: • lowering of blood pressure & decreasing CHD risk • Relaxation of cardiac muscle • Vasodilation of vascular smooth muscle • Increased excretion of sodium and water by kidney • Decreased aggregation by platelet cells

  45. cGMPThe cGMP Signal Transduction Pathway • Two forms of guanylate cyclase • Membrane-bound • Activated by ANF (atrial natriuretic factor) • ANF released when BP elevated • Cytosolic • Activated by nitric oxide • NO produced from arginine by NO synthase (activated by Ca) • Nitroglycerine slowly produces NO, relaxes cardiac and vascular smooth muscle, reduces angina • cGMP activates Protein Kinase G • Phosphorylates smooth muscle proteins

  46. cGMPThe cGMP Signal Transduction Pathway

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