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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 Interactions Substrate 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 Interactions Allosteric Regulation Binding of allosteric effectors at allosteric sites affect catalytic efficiency of the enzyme

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

9. Non-covalent Interactions Allosteric 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 Interactions Protein-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 Activity Phosphorylation and Dephosphorylation Addition or deletion of phosphate groups to particular serine, threonine, or tyrosine residues alter the enzymes activity

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

18. Covalent Regulation of Enzyme Activity Enzyme 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 Cells Intracellular 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 Receptors Ligand-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 Receptors Catalytic 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 Receptors G 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 (G s) –Activate adenylate cyclase Inhibitory G proteins (G i ) –Inhibit adenylate cyclase

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

31. Alpha proteins are different in G s and G i –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. Cholera mechanism 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 G s -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, HCO 3

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 & IP 3 Phosphotidylinositol Signal Transduction Pathway Hormone activation of phospholipase C –Via G p protein Phospholipase C hydrolyzes membrane phospholipids (phosphotidyl inositol) to produce DAG and IP 3

42. DAG & IP 3 Phosphotidylinositol Signal Transduction Pathway IP 3 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. cGMP The 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. cGMP The 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. cGMP The cGMP Signal Transduction Pathway