1. Regulation of Glycogen Metabolism Protein Kinases Protein Phosphatases cAMP G proteins Calcitonin Insulin, glucagon, and epinephrine

2. Biochemical Definitions 1. Equilibrium: (Kinetics) When the rate of the forward reaction matches the rate of the reverse reaction. Does not specify quantity at equilibrium. 2. Equilibrium constant: (Thermodynamics) The quantity [B]/[A] at equilibrium. Does not specify rate. 4. Steady-state: A dynamic condition that allows flux without changing the concentration of components in the pathway. 3. Flux: Net carbon flow in one direction A BCConsider

3. Connecting Kinetics with Thermodynamics ∆G o =  RT ln Keq Chemistry Biochemistry ∆G o =  RT ln Keq ’ pH = 7.0 (10 -7 M [H + ] )

4. Practically Speaking….. 1. A negative ∆G o ’ means [P] > [S] at equilbrium. ∆G o =  RT ln Keq ’ A B 2-1.8 10 -5.9 100 -11.9 1,000 -17.8 10,000 -23.7 100,000 -29.7 [B]/[A] ∆G o ’ (kJ/mol)

5. Steady-State vs Equilibrium Reactions Rule: An equilibrium reaction occurs in a closed system whereby two components, a reactant and a product, achieve a constancy of concentration based on chemical potentials A B Rule: A steady-state reaction occurs in an open system whereby two or more components achieve a constancy of concentration based on similar rates of components entering and leaving the system A B ZY See Strategies p 163-167 See Strategies p 163-167

6. A k1k1 k2k2 When k1k1 k2k2 A is constant = Representing a Steady-State When k1k1 k2k2 < A decreases When k1k1 k2k2 > A increases

7. Rule: A shift away from a dynamic steady-state evokes factors to restore the steady-state. Rule: Restoring steady-state requires modulating the activity of a rate-controlling enzyme(s) in the pathway Basics of Metabolic Homeostasis 1. Covalent modification 2. Changes in pathway [S] or enzyme cofactors 3. Allosterism (Vmax or Km) Enzyme activity can be modulated by: 4. Hormonal intervention 5. Enzyme turnover

8. Regulation of Carbon Flux Net carbon flux through an individual step in a pathway is defined as the difference between the forward and reverse reaction velocities J = V F - V R (these are rates of change) At equilibrium: V F = V R ; J = 0; i.e., there is no net flux even though V F and V R could be large When V F >> V R, J = V F At steady state, J = k (constant) At steady state, J depends on the rate determining (slowest) step in the pathway Net carbon flux through an individual step in a pathway is defined as the difference between the forward and reverse reaction velocities J = V F - V R (these are rates of change) At equilibrium: V F = V R ; J = 0; i.e., there is no net flux even though V F and V R could be large When V F >> V R, J = V F At steady state, J = k (constant) At steady state, J depends on the rate determining (slowest) step in the pathway Textbook p591 A B C D Rate-determining

9. Lactate Glucose Rate-controlling Step J = 0 V F > V R Meaning of Flux

10. Flux varies with equilibrium position 1.If [A] = 100mM; [B] = 10mM, an increase in [A] by 10 mM would increase flux would by 10%. (from 10:1 to 11:1) 2.If [A] = 20mM; [B] = 10mM, an increase in [A] by 10 mM would increase flux by 33%. (2:1 to 3:1) 3.If [A] = 10mM; [B] = 10mM, an increase in [A] by 10 mM would increase flux by 100% (1:1 to 2:1). A B A A A

11. AMP (0.1 mM) Adenylate Kinase2ADP = AMP + ATP Rule: Regulators with the lowest steady-state concentration have the greatest impact on regulation ATP (5 mM)ADP (1 mM) ATP + XATP + X~P + ADP 50.54.50.5 2ADP ATP + AMP 0.5 Final Talley:Before ATP5 mM After 5 mMNo change ADP1 mM No change AMP0.1 mM0.6 mM6 times

12. Why are there 2 enzymes in glycogen metabolism? Glycogen Glucose 1-PO 4 Glycogenase How can glycogen synthesis and degradation be tuned to the needs of the cell? Only glycogen or glucose 1-PO 4 concentration can affect V F or V R Glycogen Glucose 1-PO 4 UDP-Glucose Phosphorylase Synthase Because separate enzymes control synthesis and degradation, V F and V R can vary depending on the enzyme that controls the direction. This opens the way to allosteric and covalent control of glycogen Stimulating or inhibiting the enzyme cannot control direction of flux

13. cAMP Protein kinase (cAPK) Ptase Syn b Syn a ADP PO 4 ATP Phos a Phos b Ptase Pkinase PO 4 ADP ATP Protein Kinase targets of cAPK Glucagon Epinephrine Glucagon Epinephrine cAMP ATP Adenyl cyclase Textbook p586 Pkinase cAMP as a Regulator of Glycogen

14. cAMP Protein kinase (cAPK) + 4 cAMP C C RR C R R cAMP C +         C 2ATP2ADP P P Active Kinase Catalytic site Calmodulin Inactive Kinase cAPK Example would be phosphorylase b kinase Now its ready to phosphorylate phosphorylase b

15. Casein kinase II (primer) Casein kinase II (primer) P Glycogen synthase kinase 3 (GSK3) P P P Priming phosphate Inactive ATP ADP Glycogen Synthase A B PP1 H2OH2O 3P i Inactivation of Glycogen Synthase

16. Phos a Phos b PP1 Pkinase PO 4 ADP ATP Pkinase PP1 Syn b Syn a ADP PO 4 ATP Phosphoprotein Phosphatase-1 (PP1) Phosphorylase Kinase a Phosphorylase Kinase b ADP ATP PO 4 H2OH2O PP1 cAPK Remember, this is the “stripper” enzyme. It takes “off” phosphate groups on proteins

17. Insulin stimulates glycogen synthesis in liver and muscle by blocking the action of GSK3 and activating PP1 GSK3 Glycogen Synthase a Glycogen Synthase b CKII ATP 3 ATP P P P Insulin X X Glucagon epinephrine Glucose- 6-phosphate Glucose PP1 ActiveInactive 3P i Text p586

18. inactive Insulin Insulin-stimulated protein kinase Epinephrine, glucagon cAPK PP1 Glycogen G subunit + P P 2ATP 2ADP ADP ATP Glycogen synthesis stimulated, breakdown blocked Glycogen Breakdown stimulated, synthesis blocked more active PP1 Glycogen G subunit P OH PP1 Glycogen G subunit OH ATP ADP (site 2 phosphorylation) (site 1 and 2 phosphorylation) (site 1 phosphorylation) Phosphoprotein Phosphatase-1 (Muscle) Less active Inhibitor P K1 K2 Text p588

19. Insulin regulation of GSK3 Text p 587 Shut down of GSK3 by PKB

20. Summary of Catabolic Hormonal Effects on Glycogen Glucagon and Epinephrine stimulate synthesis of cAMP cAMP activates phosphorylase b kinase that converts phosphorylase b to phosphorylase a Phosphorylase a breaks down glycogen at an accelerated rate cAMP also inactivates PP1, prolonging the action of phosphorylase a

21. Summary of Anabolic Effects on Glycogen Insulin activates glycogen synthase by stimulating phosphorylation of GDK3 GDK3 inactivates itself allowing phosphoprotein phosphatase (PP1) to remove phosphate and activate glycogen synthase PPI also removes phosphate from phosphorylase a thereby shutting off glycogen breakdown

22. P Adenylcyclase CC R R cAMP dependent protein kinase     Phosphorylase kinase Phosphorylase P See Tutorial on cAMP-dependent Protein Kinases available on the web

23. Liver P P Glucose T State R State Phosphorylase a Phosphorylase b Active Low Glucose High Glucose + 5’-AMP Binds weakly to b form Phosphoprotein phosphatase-1 Low glucose a form (R state) is resistant to phosphatase P High glucose a form (T state) is vulnerable to phosphataseP Text p446-447

24. Summary of Phosphoprotein Phosphatase-1 (Liver) 1. The Phosphatase binds to phosphorylase a (T or R form) 2. The T form has a serine exposed to allow hydrolysis of -PO 4 3. The R form of phosphorylase a -PO 4 cannot be hydrolyzed 4. Glucose converts R to T form which causes hydrolysis of -PO 4 and dissociate the Phosphatase. 5. The liberated Phosphatase is free to activate glycogen synthase, thereby stimulating glycogen synthesis (It does not bind to glycogen or a G protein) (glucose is an allosteric effector of phosphorylase in liver)