CHAPTER 15 Principles of Metabolic Regulation

1. CHAPTER 15Principles of Metabolic Regulation Key topics: • Principles of metabolic regulation • Mathematical analysis of Metabolic Flux • Reciprocal regulation of Glycolysis and gluconeogenesis • Chemistry and regulation of glycogen metabolism

2. Metabolic Pathways • The biochemical reactions in the living cell — the metabolism — is organized into metabolic pathways • The pathways have dedicated purposes • Some are dedicated to extraction of energy • Some are dedicated to storage of fuels • Some are dedicated for synthesis of important building blocks • Some are dedicated to elimination of waste materials • The pathways can be represented as a map • Follow the fate of metabolites and building blocks • Identify enzymes that act on these metabolites • Identify points and agents of regulation • Identify sources of metabolic diseases

3. Map of Metabolic Pathways http://www.genome.jp/kegg/pathway/map/map01100.html Generic Metabolic map Organism specific maps Functional Enzyme Nomenclature Links to Sequence Databases Genome =>Transcriptome => Proteome => Metabolome

4. Homeostasis • Organisms maintain homeostasis by keeping the concentrations of most metabolites at steady state • After brief adaptation, single-celled organisms (yeast and bacteria) exhibit balanced, exponential, steady-state growth where molecular proportions are maintained over large ranges of cell density • In steady state, the rate of synthesis of a metabolite equals the rate of breakdown of this metabolite

5. Factors that Determine the Activity of Enzymes

6. Active Protein Molecules have a Finite Lifespan • Different proteins in the same tissue have very different half-lives • less than an hour to about a week for liver enzymes • Stability determined by sequence, structure actively managed by ubiquitin / proteasome system • Steady state is when production is balanced by decay + dilution

7. Phosphorylation of Enzymes Affects their Activity • Protein phosphorylation is catalyzed by protein kinases • Dephosphorylation is spontaneous, or catalyzed by protein phosphatases • Typically, hydroxyl groups of Ser, Thr, or Tyr are phosphorylated

8. Michaelis Menten Kinetics -- simplest case for substrate effects Reaction rates depend on substrate concentrations according to enzyme binding and turnover characteristics

9. AMP is a sensitive indicator of Cellular Energy Capacity

10. AMP-activated protein kinase (AMPK) global mobilization of energy stores

11. 15.1 Regulation of Metabolic Pathways Steady State = balanced production and consumption of intermediates perturbations accommodated by feedback Regulation Cells modify metabolic flux on short (ms) and Long (days) time scales Signals modify trx => mRNA (transcriptome) => protein (proteome) => intermediates (metabolome) Regulatory steps far from equilibrium ATP NADH nearly constant ATP/AMP reflects energy status - tightly modulated.

12. 15.2 Analysis • Flux control distributed among several enzymes • C (Flux control coefficient) effect of 1 enzyme on overall flux • ε elasticity coefficient V/S • R response coefficient • R = C *ε • Flux toward a product most effectively increase by raising all enzymes

13. Some Enzymes in the Pathway Limit the Flux of Metabolites More than Others • Addition of pure enzyme to Glycolytic homogenate • Few enzymes limit overall Flux • Increased hexokinase activity enables activation of glucose • Increased phosphofructokinase-1 activity enables catabolism of activated glucose via glycolysis

14. Reactions Far From Equilibrium are Common Points of Regulation • Allosteric effectors or protein modifications cannot modify the free energy difference between substrates and products, • Only absolute rates, not relative rates • Net flux through this pathway is 10 • First step most sensitive to modulation

15. Rates of a Biochemical Reactions • Rates of a biochemical reactions depend on many factors • Concentration of reactants • Activity of the catalyst • Concentration of the enzyme • Intrinsic activity of the enzyme • Concentrations of effectors • Allosteric regulators • Competing substrates • pH, ionic environment • Temperature

16. Elasticity Coefficient Measures the Responsiveness to Substrate ε elasticity coefficient ε = Vo/S

17. 15.3 Coordinated Regulation of Glycolysis and Gluconeogenesis • 7 Es shared, 3 other steps points of regulation • Hexokinase IV liver • PFK-1 inhibited by ATP and citrate • Pyruvate Kinase inhbited by cAMP and by phosphorylation in liver • Gluconeogenesis regulated by Pyruvate carboxylase (acetyl-coa) and FBPase (F26bp and AMP) • Glcneo and Glcl under reciprocal control to limit futile cycling • Glucagon and epinephrine decrease F26BP by raising cAMP;PFK-2/FBPase-2 gets phosphorylated • Insulin increases F26BP by activating a P-Protein phosphatases- X5P activates PP2a dephosphorylates PFK2P => PFK2; • glucose uptake^, glycogen^, lipid synth^ • Trx factors ChREBP, CREB, SREBP and FOXO1 regulate metabolic genes in response to Insulin and Glucagon

18. Glycolysis vs. Gluconeogenesis

19. Regulation of Hexokinase IV by Sequestration

20. Isozymes may Show Different Kinetic Properties • Isozymes are different enzymes that catalyze the same reaction • They typically share similar sequences • Their regulation is often different

21. Regulation of Phosphofructokinase-1 • The conversion of fructose-6-phosphate to fructose 1,6-bisphosphate is the commitment step in glycolysis • ATP is a negative effector • Do not spend glucose in glycolysis if there is plenty of ATP

22. Regulation of Phosphofructokinase 1 and Fructose 1,6-Bisphosphatase • Gluconeogenesis if AMP is low • Glycolysis if AMP is high and ATP is low

23. Regulation by Fructose 2,6-Bisphosphate • F26BP activates phosphofructokinase (glycolytic enzyme) • F26BP inhibits fructose 1,6-bisphosphatase (gluconeogenetic enzyme)

24. Regulation by Fructose 2,6-Bisphosphate Gluconeogenesis if F26BP is low Glycolysis if F26BP is high

25. Regulation of 2,6-Bisphosphate Levels

26. Molecular Origin of Enzyme Regulation • Regulation of catalysis typically involves • Binding of inhibitors, often to the active site • Binding of regulatory protein subunits

27. Regulation of Pyruvate Kinase • Signs of abundant energy supply allosterically inhibit all pyruvate kinase isoforms • Signs of glucose depletion (glucagon) inactivate liver pyruvate kinase via phosphorylation • Glucose from liver is exported to brain and other vital organs

28. Two Alternative Fates for Pyruvate • Pyruvate can be a source of new glucose • Store energy as glycogen • Generate NADPH via pentose phosphate pathway • Pyruvate can be a source of acetyl-CoA • Store energy as body fat • Make ATP via citric acid cycle • Acetyl-CoA stimulates glucose synthesis by activating pyruvate carboxylase

29. Trx factors ChREBP and FOXO1 regulate metabolic genes in response to Insulin and Glucagon

31. Glycogen Metabolism

32. Control of Glycogen Synthesis • Insulin signaling pathway • increases glucose import into muscle • stimulates the activity of muscle hexokinase • activates glycogen synthase • Increased hexokinase activity enables activation of glucose • Glycogen synthase makes glycogen for energy storage

33. Branch Points in Glycogen • Glycogen phosphorylase works on non-reducing ends until it reaches four residues from an (1 6) branch point • Debranching enzyme transfers a block of three residues to the non-reducing end of the chain • Debranching enzyme cleaves the single remaining (1 6) –linked glucose

34. Epinephrine and Glucagon Stimulate Breakdown of Glycogen

35. Chapter 15: Summary • living organisms regulate the flux of metabolites via metabolic pathways by • increasing or decreasing enzyme concentrations • activating or inactivating key enzymes in the pathway • the activity of key enzymes in glycolysis and gluconeogenesis is tightly regulated via various activating and inhibiting metabolites • glycogen synthesis and degradation is regulated by hormones insulin, epinephrine, and glucagon that report on the levels of glucose in the body