1. Biochemistry 432/832 September 03 Chapter 23 G&G Gluconeogenesis Glycogen metabolism

2. Announcements: -

3. Comparison of glycolysis and gluconeogenesis pathways

4. Energetics of Glycolysis The elegant evidence of regulation!  G in cells is revealing: Most values near zero 3 of 10 reactions have large, negative  G Large negative  G reactions are sites of regulation! Reactions 1, 3 and 10 should be different to go into opposite direction

5. Gluconeogenesis Something Borrowed, Something New Seven steps of glycolysis are retained: –Steps 2 and 4-9 Three steps are replaced: –Steps 1, 3, and 10 (the regulated steps!) The new reactions provide for a spontaneous pathway (  G negative in the direction of sugar synthesis), and they provide new mechanisms of regulation

6. Pyruvate Carboxylase Pyruvate is converted to oxaloacetate The reaction requires ATP and bicarbonate as substrates Biotin-dependent Biotin is covalently linked to an active site lysine Acetyl-CoA is an allosteric activator Regulation: when ATP or acetyl-CoA are high, pyruvate enters gluconeogenesis The "conversion problem" in mitochondria

7. The pyruvate carboxylase reaction

8. Linkage of biotin to lysine residue in pyruvate carboxylase

9. Pyruvate carboxylase is a compartmentalized enzyme Oxaloacetate is formed in mitochondria It cannot be transported to the cytosol It is converted to malate in mitochondria and back to oxaloacetate in the cytosol

10. PEP Carboxykinase Conversion of oxaloacetate to PEP Lots of energy needed to drive this reaction! Energy is provided in 2 ways: –Decarboxylation is a favorable reaction –GTP is hydrolyzed GTP used here is equivalent to an ATP

11. The PEP carboxykinase reaction

12. Fructose-1,6-bisphosphatase Hydrolysis of F-1,6-P to F-6-P Thermodynamically favorable -  G in liver is -8.6 kJ/mol Allosteric regulation: –citrate stimulates –fructose-2,6-bisphosphate inhibits –AMP inhibits

13. The fructose-1,6- biphosphatase reaction

14. Glucose-6-Phosphatase Conversion of Glucose-6-P to Glucose Presence of G-6-Pase in ER of liver and kidney cells makes gluconeogenesis possible Muscle and brain do not do gluconeogenesis G-6-P is hydrolyzed as it passes into the ER ER vesicles filled with glucose diffuse to the plasma membrane, fuse with it and open, releasing glucose into the bloodstream

15. Glucose-6-phosphatase is localized in the ER

16. Lactate Recycling How your liver helps you during exercise.... Vigorous exercise can lead to a buildup of lactate and NADH, due to oxygen shortage and the need for more glycolysis NADH can be reoxidized during the reduction of pyruvate to lactate Lactate is then returned to the liver, where it can be reoxidized to pyruvate by liver LDH Liver provides glucose to muscle for exercise and then reprocesses lactate into new glucose

17. The Cori Cycle

18. Gerty and Carl Cori

19. Cori Cycles

20. Regulation of Gluconeogenesis Reciprocal control with glycolysis When glycolysis is turned on, gluconeogenesis should be turned off When energy status of cell is high, glycolysis should be off and pyruvate, etc., should be used for synthesis and storage of glucose When energy status is low, glucose should be rapidly degraded to provide energy The regulated steps of glycolysis are the very steps that are regulated in the reverse direction!

21. Regulation of glycolysis and gluconeogenesis - - - - - - - - - + + + +

22. Gluconeogenesis Regulation II Allosteric and Substrate-Level Control Glucose-6-phosphatase is under substrate- level control, not allosteric control The fate of pyruvate depends on acetyl-CoA F-1,6-bisPase is inhibited by AMP, activated by citrate - the reverse of glycolysis Fructose-2,6-bisP is an allosteric inhibitor of F-1,6-bisPase

23. Inhibition of fructose-1,6-bisphosphatase by fructose-2,6-bisphosphate - synergistic effect of F-2,6-P and AMP No AMP 25  M AMP F-2,6-PAMP F-2,6-P

24. Synthesis and degradation of F-2,6,-bisP are catalyzed by the same enzyme + -

25. Substrate cycles -

26. Simultaneous activity of Phosphofructokinase (glycolysis) and F-1,6-bisPase (gluconeogenesis) yields a substrate cycle Reverse reaction decreases steady state flux through the pathway Could explain how 10% change in ATP concentration results in ~90-fold increase in the flux through glycolysis Synergistic Fructose-2,6-bisP / AMP / ATP / citrate (NAD + /NADH ratio, glucose-6-P, pyruvate, etc.) regulation provide alternative explanation

27. Substrate cycles Three potential substrate cycles in glycolysis and gluconeogenesis Example of Phosphofructokinase (glycolysis) and F-1,6-bisPase (gluconeogenesis) Reciprocal regulation does not work at high [F-1,6-P] Perhaps substrate cycling occurs only at high concentrations of F-1,6-P (PFK product) - - this prevents accumulation of excessively high levels of F-1,6-P

28. Glucose Fructose-6-P Glucose-6-P Glyceraldehyde-3-P Pyruvate ATP GlycogenRibose-5-P + NADPH Nucleic acid synthesis Reducing power

29. 23.3 Glycogen Catabolism Getting glucose from storage (or diet) ·Glycogen is a storage form of glucose  -Amylase is an endoglycosidase It cleaves amylopectin or glycogen to maltose, maltotriose and other small oligosaccharides It is active on either side of a branch point, but activity is reduced near the branch points Debranching enzyme cleaves "limit dextrins" The 2 activities of the debranching enzyme

30. Hydrolysis of glycogen by amylases

31. The reactions of glycogen debranching enzyme. 1) Transfer of 3 glucose residues to another branch and 2) cleavage of a single glucose residue at the branch point

32. Metabolism of Tissue Glycogen Digestive breakdown is unregulated - 100%! But tissue glycogen is an important energy reservoir - its breakdown is carefully controlled Glycogen consists of "granules" of high MW Glycogen phosphorylase cleaves glucose from ends of glycogen molecules This is a phosphorolysis, not a hydrolysis Metabolic advantage: product is a sugar-P - a "sort- of" glycolysis substrate

33. The glycogen phosphorylase reaction - phosphorolysis

34. Glycogen Phosphorylase A beautiful protein structure! A dimer of identical subunits (842 res. each) Each subunit contains a PLP, which participates in phosphorolysis Chapter 15

35. Dimer Monomer