1. Glycogen Metabolism and Gluconeogenesis CH 339K

2. Glycolysis (recap) We discussed the reactions which convert glucose to pyruvate: C 6 H 12 O 6 +2 NAD + + 2 ADP  2 CH 3 COCOOH + 2 NADH +2 ATP + 2 H + What about the sources of glucose? –Dietary sugars –Glycogen

3. Before we get to glycogen: Dietary sugars

4. Amylase Reaction

5. Glycogen Branched every 8-12 residues Up to 50,000 or so residues total

6. Breakdown: Glycogen Phosphorylase

7. Glycogen Synthesis and Breakdown Glycogen synthesis and breakdown are both controlled by hormones Glucagon, Epinephrine –turn on glycogen breakdown –Turn off glycogen synthesis Hormones act through receptors on cell surface and G-proteins Glucagon – 29 amino acid polypeptide produced in pancreas in response to low blood sugar Epinephrine – aka adrenaline – produced by adrenal medulla in response to stress

8. Activation of Glycogen Phosphorylase 3’-5’ cyclic AMP G-Proteins Second messengers Kinase Cascade

9. G-Proteins G proteins are heterotrimers, containing G , G  and G  subunits. SubunitSize GaGa45 – 47 kD GG 35 kD GgGg7-9 kD

10. G-Proteins The G  subunits bind guanine nucleotides (hence the name “G Protein”). G Proteins are associated on one hand with the inner surface of the plasma membrane, and on the other hand with membrane spanning receptor proteins called G-protein coupled receptors or GPCRs. There are a number of different GPCRs; most commonly these are receptors for hormones or for some type of extracellular signal. In the “resting” state, G  is bound to the G  -G  dimer. G  contains the nucleotide binding site, holding GDP in the inactive form, and is the “warhead” of the G protein. At least 20 different forms of Ga exist in mammalian cells. Binding of the extracellular signal by the GPCR causes it to undergo an intracellular conformational change; this causes an allosteric effect on G . The change in G  causes it to exchange GDP for GTP. GTP activates G , causing it to dissociate from the G  -G  dimer. The activated G  binds and activates an effector molecule. G  also has a slow GTPase activity. Hydrolysis of GTP deactivates G , which reassociates with the G  -G  dimer and the GPCR to reform the resting state. In other words, G-protein mediated cellular responses have a built-in off switch to prevent them from running forever.

11. G-Protein Coupled Receptors (GPCRs)

12. G-Proteins – Effect of GDP/GTP Binding GTP – terminal PO 4 constrains the  -binding loop (red) GDP – missing terminal PO 4 allows the  -binding loop (red) to assime a looser conformation

13. Cycling of G protein between active and inactive states

14. G-Protein Killers Cholera Cholera toxin secreted by the bacterium Vibrio cholera. A subunit and five B subunits. A subunit catalyzes the transfer of an ADP-ribose from NAD+ to a specific Arg side chain of the α subunit of Gs. G  is irreversibly modified by addition of ADP-ribosyl group; Modified Gα can bind GTP but cannot hydrolyze it ). As a result, there is an excessive, nonregulated rise in the intracellular cAMP level (100 fold or more), which causes a large efflux of Na+ and water into the gut. Pertussis (whooping cough) Pertussis toxin (secreted by Bordetella pertussis) catalyzes ADP-ribosylation of a specific cysteine side chain on the α subunit of a G protein which inhibits adenyl cyclase and activates sodium channels. This covalent modification prevents the subunit from interacting with receptors; as a result, locking Gα in the GDP bound form. You probably vaccinate your dog against the related species that causes kennel cough.

15. Cholera is still a problem- 2009 Zimbabwe outbreak – 4300 deaths

16. Activation of Adnylate Cyclase

17. Activation of cAMP-Dependant Protein Kinase

18. Glycogen Phosphorylase Exists in 2 forms –Phosphorylase B (inactive) –Phosphorylase A (active) Phosphorylase B is converted to Phosphorylase A when it is itself phosphorylated by Synthase Phosphorylase Kinase (SPK) GP cannot remove branch points (  -1,6 linkages)

19. Activation of Glycogen Phosphorylase 3’-5’ cyclic AMP cAMP – dependent Protein Kinase

20. Activation of Glycogen Phosphorylase cAMP – dependent Protein Kinase PLP: Pyridoxal Phosphate cofactor

21. Debranching Enzyme The activity of phosphorylase ceases 4 glucose residues from the branch point. Debranching enzyme (also called glucan transferase) contains 2 activities: –glucotransferase –glucosidase. Glycogenolysis occurring in skeletal muscle could generate free glucose which could enter the blood stream. However, the activity of hexokinase in muscle is so high that any free glucose is immediately phosphorylated and enters the glycolytic pathway.

22. Cori Disease Cori disease (Glycogen storage disease Type III) is characterized by accumulation of glycogen with very short outer branches, caused by a flaw in debranching enzyme. Deficiency in glycogen debranching activity causes hepatomegaly, ketotic hypoglycemia, hyperlipidemia, variable skeletal myopathy, cardiomyopathy and results in short stature.

23. Glycogen Synthesis Glycogen Synthase adds glucose residues to glycogen Synthase cannot start from scratch – needs a primer Glycogenin starts a new glycogen chain, bound to itself

24. Glycogen Synthesis (cont.) Synthase then adds to the nonreducing end.

25. Glycogen Synthesis (cont.) To add to the glycogen chain, synthase uses an activated glucose, UDP- Glucose UDP-Glucose Pyrophosphorylase links UDP to glucose

26. Glycogen Synthesis (cont.) Synthase then adds the activated glucose to the growing chain Release and subsequent hydrolysis of pyrophosphate drives the reaction to the right

27. Glycogen Synthesis (cont.) Glycogen branching enzyme then introduces branch points

28. Mature Glycogen Built around glycogenin core Multiple non- reducing ends accessible to glycogen phosphorylase

29. Reverse Regulation of Phosphorylase and Synthase The same kinase phosphorylates both glycogen phosphorylase and synthase Synthase I (dephos.) is always active Synthase D (phos.) is dependent on [G-6-P] The same event that turns one on turns the other one off.

30. Gluconeogenesis CH 339K

31. Gluconeogenesis Average adult human uses 120 g/day of glucose, mostly in the brain (75%) –About 20g glucose in body fluids –About 190 g stored as glycogen –Less than 2 days worth In addition to eating glucose, we also make it Mainly occurs in liver (90%) and kidneys (10%) Not the reverse of glycolysis Differs at the irreversible steps in glycolysis

32. Gluconeogenesis Differs Here And Here

33. First Difference Glycolysis: make a nucleotide triphosphate Gluconeogenesis: burn two nucleotide triphosphates

34. Pyruvate Carboxylase

35. PEP Carboxykinase

36. Malate Shuttle Pyruvate Carboxylase is mitochondrial OAA reduced to malate in matrix Carrier transports malate to cytoplasm Cytoplasmic malate dehydrogenase reoxidizes to OAA Mammals have a mitochondrial PEPCK

37. Second and Third differences

38. Energetics Gluconeogenesis Pyruvate + 4 ATP + 2 GTP + 2 NADH + 2 H 2 O ⇌ glucose + 4 ADP + 2 GDP + 2 NAD+  G = -37 kJ/mol Glycolysis (reversed) Pyruvate + 2 ATP + 2 NADH + 2 H 2 O ⇌ glucose + 2 ADP + 2 NAD+  G = +84 kJ/mol Net difference of 4 nucleotide triphosphate bonds at ~31 kJ each accounts for difference in  Gs

39. Local Regulation Phosphofructokinase-1(Glycolysis) is inhibited by ATP and Citrate and stimulated by AMP. Fructose-1,6-bisphosphatase (Gluconeogenesis) is inhibited by AMP.

40. Global Control Enzymes relevant to these pathways that are phosphorylated by cAMP-Dependent Protein Kinase include: Pyruvate Kinase, a glycolysis enzyme that is inhibited when phosphorylated. A bi-functional enzyme that makes and degrades an allosteric regulator, fructose- 2,6-bisphosphate.

41. Pyruvate Kinase Regulation Local regulation by substrate activation Global regulation by hormonal control of Protein Kinase A

42. Effects of Fructose-2,6-Bisphosphate Fructose-2,6-bisphosphate allosterically activates the glycolysis enzyme Phosphofructokinase-1, promoting the relaxed state, even at relatively high [ATP]. Activity in the presence of fructose- 2,6-bisphosphate is similar to that observed when [ATP] is low. Thus control by fructose-2,6-bisphosphate, whose concentration fluctuates in response to external hormonal signals, supercedes control by local conditions (ATP concentration). Fructose-2,6-bisphosphate instead inhibits the gluconeogenesis enzyme Fructose-1,6-bisphosphatase.

43. Source of Fructose-2,6-Bisphosphate Fructose-2,6-bisphosphate is synthesized and degraded by a bi- functional enzyme that includes two catalytic domains Phosphofructokinase-2 (PFK2) domain catalyzes: fructose-6-phosphate + ATP ⇄ fructose-2,6-bisphosphate + ADP. Fructose-Biosphosphatase-2 (FBPase2) domain catalyzes: fructose-2,6-bisphosphate + H2O ⇄ fructose-6-phosphate + Pi. Phosphorylation activates FBPase2 and inhibits PFK2

44. BifunctionalEnzyme Activates PFK1 Inhibits F-1,6-bisphosphatase Inhibits PFK1 Activates F-1,6-bisphosphatase

45. Reciprocal Regulation of PFK-1 and FBPase-1

46. Medical aside – nonlethal! People with Type II diabetes have very high (~3x normal) rates of gluconeogenesis Initial treatment is usually with metformin. Metformin shuts down production of PEPCK and Glucose-6-phosphatase, inhibiting gluconeogenesis.

47. Futile Cycles Occur when loss of reciprocal regulation fails twixt glycolysis and gluconeogenesis Anesthestics like halothane occasionally lead to runaway cycle between PFK and fructose-1,6-BPase Malignant Hyperthermia

48. The Cori Cycle High NADH/NAD + Low NADH/NAD +