Regulation of Metabolism

1. Regulation of Metabolism FCSN 543 Advanced Nutritional Biochemistry Dr. David L. Gee

2. 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

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

4. 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

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

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

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

8. 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

9. 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

10. 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

11. 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

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

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

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

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

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

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

18. 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

19. 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

20. Hormones, Receptors, and Communication Between CellsIntracellular Receptors

21. 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

22. Hormones, Receptors, and Communication Between Cells • 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

23. Hormones, Receptors, and Communication Between CellsCell-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

24. Hormones, Receptors, and Communication Between CellsCell-Surface Receptors • G-protein-linked receptors • Binding of hormone activates an enzyme via a G-protein communication link. • The enzymes produces intracellular messengers • cAMP • diacylglycerol (DAG))

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

26. 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

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

28. 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

29. 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)

30. 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

31. 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

32. DAG & IP3Phosphotidylinositol Signal Transduction Pathway • Protein kinase C activated by DAG and calcium • Synthesis of DAG and IP3

33. 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

34. 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 • Nitroglycerine slowly produces NO, relaxes cardiac and vascular smooth muscle, reduces angina • cAMP activates Protein Kinase G • Phosphorylates smooth muscle proteins

35. cGMPThe cGMP Signal Transduction Pathway