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Glycolysis, concluded; Gluconeogenesis

Glycolysis, concluded; Gluconeogenesis. Andy Howard Biochemistry Lectures, Fall 2010 20 October 2010. Glycolysis Pyruvate Free energy Regulation Other sugars Entner-Doudoroff Pathway. Gluco-neogenesis. What we’ll discuss. Glycolytic pathway through TIM. Figure Courtesy U.Texas.

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Glycolysis, concluded; Gluconeogenesis

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  1. Glycolysis, concluded; Gluconeogenesis Andy HowardBiochemistry Lectures, Fall 201020 October 2010 Glycolysis II, Gluconeogenesis I

  2. Glycolysis Pyruvate Free energy Regulation Other sugars Entner-Doudoroff Pathway Gluco-neogenesis What we’ll discuss Glycolysis II, Gluconeogenesis I

  3. Glycolytic pathway through TIM Figure Courtesy U.Texas Glycolysis II, Gluconeogenesis I

  4. Final steps Bottom half of same graphic Glycolysis II, Gluconeogenesis I

  5. Concentrations of glycolytic metabolites in erythrocytes Metabolite Conc,mM Metabolite Conc,mM Glucose 5.0 G-6-P 0.083 F-6-P 0.014 F-1,6-bisP 0.031 DHAP 0.14 Glyc-3-P 0.019 1,3-bisPglyc 0.001 2,3-bisPglycate 4.0 3-P-glycate 0.12 2-P-glycate 0.030 PEP 0.023 Pyruvate 0.051 Lactate 2.9 ATP 1.85 ADP 0.14 Phosphate 1.0 Glycolysis II, Gluconeogenesis I

  6. Hexokinase • Transfers γ-phosphoryl group of ATP to oxygen atom at C-6 of glucose, producing glucose 6-phosphate and ADP. • Coupling between ATP hydrolysis and an energy-requiring reaction is very close: phosphate is transferred directly from ATP to the recipient molecule, in this case glucose. • Reaction catalyzed by hexokinase is energetically favored: Go’ = -22.3 kJ/mol Glycolysis II, Gluconeogenesis I

  7. Hexokinase isozymes • various isozymes (functionally related but structurally slightly distinct) forms of hexokinase in humans • liver form (glucokinase -- see next slide) has Km in millimolar range, perhaps a factor of 1000 higher than the Km of hexokinase found in other tissue • Liver form is therefore much less active than the other forms unless the liver glucose concentration is high Glycolysis II, Gluconeogenesis I

  8. Activity&complexity • Hexokinase is active on sugars besides glucose;activity against maltose is comparable to the activity on glucose • Hexokinase has the highest molecular mass per monomer of any of the glycolytic enzymes;given that it is the first enzyme in an important pathway,it makes sense that it is large and complex. Human glucokinasePDB 3F9M 52kDa monomer1.5Å; EC 2.7.1.2 Glycolysis II, Gluconeogenesis I

  9. Phosphoglucomutase(phosphoglucose isomerase) • Interconverts phosphorylated forms of glucose—glucose 1-P and glucose 6-P. • Intermediate is bisphosphorylated • equilibrium between the 1-P and 6-P forms is determined by relative concentrations. Glycolysis II, Gluconeogenesis I

  10. Phospho-glucomutase, cont’d • Active on other phosphorylated aldoses in addition to glucose. • This enzyme doesn’t appear on the chart:not part of the linear pathway from glucose to pyruvate. • But it’s relevant in various contexts L.lactis Phospho-glucomutasePDB 1O0825 kDa monomer1.2Å; EC 5.4.2.6 Glycolysis II, Gluconeogenesis I

  11. Glucose 6-P isomerase(phosphohexose isomerase) • Interconverts glucose 6-P & fructose 6-phosphate. • Proceeds through (1,2) ene-diol intermediate • with enzyme present the energy barriers around this ene-diol are lowered enough to speed the interconversion. Glycolysis II, Gluconeogenesis I

  12. Properties of G6P isomerase • Dimeric enzyme plays roles extracellularly as well as intracellularly: it can function as a nerve growth factor. • Each monomer contains two unequal-sized domains, and the active site is formed by the association of the two subunits. S.aureusG6P isomerase101 kDa dimer(monomer shown) 1.65Å EC 5.3.1.9 Glycolysis II, Gluconeogenesis I

  13. Phosphofructokinase-1 • catalyzes phosphorylation at the 1 position of fructose 6-phosphate. • example of a kinase that acts on an already-phosphorylated form, creating a bisphosphorylated compound. • ADP acts as an allosteric activator on this enzyme as well as being a product of the reaction. Glycolysis II, Gluconeogenesis I

  14. PFK-1 structures • Several recent determinations as parts of structural genomics projects • Most are tetramers • Some are less allosteric than others (this one is just barely allosteric, whereas the E.coli enzyme is distinctly so) L.bulgaricus PFK-1137 kDa tetramerPDB 1ZXX 1.85Å; EC 2.7.1.11 Glycolysis II, Gluconeogenesis I

  15. F-1,6-bisP aldolase • Catalyzes actual C-C bond cleavage: F1-6 DHAP + glyceraldehyde-3-P • Large, important enzyme • Some bacterial and yeast forms require a divalent cation as a cofactor; eukaryotic aldolases do not. • The non-cationic forms proceed through an imine (Schiff-base) intermediate. M.tuberculosisaldolase151 kDa tetramermonomer shownPDB 3ELF1.31Å; EC 4.12.13 Glycolysis II, Gluconeogenesis I

  16. Secondary activity F-1-P • Enzyme is active on fructose 1-phosphate as well as its "standard" substrate, fructose 1,6-bisphosphate • in this context it forms part of catabolic pathway by which fructose itself can be used as an energy and carbon source. F-1,6 bisP Glycolysis II, Gluconeogenesis I

  17. Triosephosphate isomerase • Interconverts two 3-C phosphosugars: dihydroxyacetone P  glyceraldehyde 3-P • possibly the most efficient enzyme known, in terms of the rate acceleration afforded by the enzyme relative to the uncatalyzed reaction. Glycolysis II, Gluconeogenesis I

  18. TIM and TIM barrels • TIM is a tetrameric enzyme with a characteristic structure in which alpha helical stretches alternate with beta strands such that the beta strands curve around to form a barrel-like structure with the helices outside. • This structural motif appears in many other enzymes, and has become known as a "TIM barrel." Leishmania TIM56 kDa dimermonomer shown PDB 2VXN 0.82Å; EC 5.3.1.1 Glycolysis II, Gluconeogenesis I

  19. Phosphoglycerate kinase • catalyzes dephosphorylationof 1,3-bisphosphoglycerateto form 3-phosphoglycerate • named for reaction runningin opposite direction relativeto the one shown in chart. • In the direction shown in table it produces ATP rather than consuming it. Glycolysis II, Gluconeogenesis I

  20. PGK Structural Notes • Has a hinge motion about a point near the center of the molecule; the open and closed forms of the enzyme involve movements as large as 17Å in the residues farthest from the hinge point. • Enzyme is primarily alpha-helical in conformation. T.thermophilus PGKPDB 1V6S 84 kDa dimer;monomer shown1.5Å; EC 2.7.2.3 Glycolysis II, Gluconeogenesis I

  21. Phosphoglycerate mutase • interconverts 3-phosphoglycerate and 2-phosphoglycerate • Mechanism of reaction involves formation of 2,3-bisphosphoglycerate via transient phosphorylation of a histidine residue of the enzyme. Glycolysis II, Gluconeogenesis I

  22. PG Mutase:A problem! • 2,3BPG can diffuse from phosphoglycerate mutase, however, leaving the enzyme trapped in an unusable state. • Cells make excess 2,3BPG (using the enzyme bisphosphoglycerate mutase) in order to drive 2,3BPG back to phosphoglycerate mutase, so the reaction can go to completion. E.coli PGMPDB 1E5857 kDa dimermonomer shown1.25Å; EC 5.4.2.1 Glycolysis II, Gluconeogenesis I

  23. Enolase • converts 2-phosphoglycerate to phosphoenolpyruvate • This reaction plays a role in gluconeogenesis as well as glycolysis. Glycolysis II, Gluconeogenesis I

  24. Enolase details • Mg2+ ions are required for activity in some forms of the enzyme. • Vertebrate genes code for two slightly different forms of the monomer of enolase, alpha and beta (and gamma…) • Most of the enolase in fetal tissue is alpha-alpha; mature skeletal muscle contains beta-beta; some alpha-alpha remains in smooth muscle tissue. Human -enolase96 kDa dimerPDB 2AKZ1.36Å; EC 4.2.1.11 Glycolysis II, Gluconeogenesis I

  25. Pyruvate kinase • transfers a phosphate from phosphoenolpyruvate to ADP, producing pyruvate and ATP • The reaction is essentially irreversible Glycolysis II, Gluconeogenesis I

  26. Regulation of Pyr kinase • Fructose 1,6-bisphosphate, the substrate for the aldolase reaction, is a feed-forwardactivator of the reaction Human M2 pyruvate kinase244 kDa tetramerPDB 3GR41.6Å; EC 2.7.1.40 Glycolysis II, Gluconeogenesis I

  27. What happens to pyruvate? • Four fates: • Pyruvate + HS–CoA + NAD Acetyl CoA + NADH + H+ (13.1) • Pyruvate + HCO3- + ATP Oxaloacetate + ADP + Pi (12.1B) • Pyruvate + H+ Acetaldehyde + CO2Acetaldehyde + NADH + H+Ethanol + NAD+ (11.3A) • Pyruvate + NADH + H+ Lactate + NAD+(11.3B) Glycolysis II, Gluconeogenesis I

  28. Glycolysis and energy • General principle:For a reaction to move forward, G < 0. • That doesn’t necessarily mean Go < 0. • See fig. 11.1: • Irreversible steps 1,3,10 • Other steps have G ≈ 0,so they’re reversible Glycolysis II, Gluconeogenesis I

  29. G and Go Glycolysis II, Gluconeogenesis I

  30. Regulation of glycolysis • Thoroughly studied • Recently it’s been recognized that a compound isn’t really an effector if it modulates a reaction only when it’s much more concentrated than it ever gets in the cell • So we need to know concentrations! Glycolysis II, Gluconeogenesis I

  31. What does the cell want? • Glycolysis should be turned on when ATP is needed • Usual arguments regarding products and downstream products • See fig. 11.12 • Hexokinase is product-inhibited • PFK1 has up & down effectors • Pyruvate kinase is: • feed-forward activated by F-1,6-bisP • Inhibited by ATP Glycolysis II, Gluconeogenesis I

  32. Regulation of transporters • Can’t metabolize glucose in a cell that doesn’t contain any glucose! • [glucose]in cell < [glucose]blood so transporters are passive • SGLT1 in intestine & kidney—see ch. 9 Diagram courtesy Steve Cook Glycolysis II, Gluconeogenesis I

  33. GLUT transporters • Six GLUT transporters with particular tasks • Insulin turns on glucose uptake into skeletal and heart muscle cells via GLUT4 (fig.11.13) Glycolysis II, Gluconeogenesis I

  34. Diagram courtesy Kobe University GLUT4 activation Glycolysis II, Gluconeogenesis I

  35. Other GLUT transporters • Basal levels maintained by GLUT1, 3 • GLUT2 moves glucose freely in and out of liver cells:therefore [glucose]cell = [glucose]blood in that particular organ • GLUT5 specific to small intestine • GLUT7: G6P from cytoplasm into ER • Hexokinase acts quickly on glucose once it’s intracellular: G6P can’t pass outward Glycolysis II, Gluconeogenesis I

  36. Hexokinase regulation • First step and it’s irreversible • Product inhibits hexokinase I,II, III but not hexokinase IV (=glucokinase in liver, islet-cells of pancreas) • Makes sense: remember [glucose] high in that environment Glycolysis II, Gluconeogenesis I

  37. Glucokinase regulation • There’s a glucokinase regulatory protein (GKRP) that binds to glucokinase in the presence of fructose-6-P and converts its hyperbolic kinetics into sigmoidal • Raises apparent Km of glucokinase • Glucokinase turned on after a meal because F1P relieves inhibition then Guessed structure of GKRP based on MODBASEmodel from G-6-P isomerase Glycolysis II, Gluconeogenesis I

  38. PFK-1 regulation by ATP, AMP • ATP is substrate and allosteric inhibitor:Increases Kmeff for F6P • AMP relieves inhibition by ATP • Effect of ADP depends on species • AMP is primary modulator because its concentration varies more than ATP Glycolysis II, Gluconeogenesis I

  39. Other modulators of PFK-1 • Citrate inhibits: if [citrate] is high, then TCA cycle is probably blocked, so there’s no point in continuing making pyruvate • pH matters: PFK-1 inhibited by low pH, so after exercise, PFK-1 activity decreases • Fructose 2,6 bisphosphate is a micromolar activator of PFK1…it’s produced from F6P by action of PFK-2 Glycolysis II, Gluconeogenesis I

  40. F-2,6-BP as regulator • Potent allosteric regulatory molecule. • activates phosphofructokinase-1. • inhibits fructose-1,6-bisphosphatase (enzyme in gluconeogenesis). • Its synthesis and degradation are catalyzed by the same bifunctional enzyme Glycolysis II, Gluconeogenesis I

  41. 6-phosphofructo-2-kinase (PFK-2) /fructose-2,6-bisphosphatase Glycolysis II, Gluconeogenesis I

  42. Low glucose Regulation of PFK-2 /fructose-2,6-bisphosphatase High glucagon • Phosphorylation of the enzyme results in the inactivation of the phosphofructokinase-2 activity and activation of the fructose-2,6-bisphosphatase activity. This results in a down regulation of glycolysis and increased gluconeogenesis. Increased phosphorylation Glycolysis II, Gluconeogenesis I

  43. PFK-2 and glucagon • In liver: low blood [glucose] triggers release of glucagon • Glucagon activates adenylyl cyclase signaling in liver cells; that enables protein kinase A to phosphorylate a ser in PFK-2 • That turns on the fructose 2,6-bisphosphatase activity and turns off PFK-2 activity • Thus [F-2,6-bisP] drops so PFK-1 activity drops • See fig. 11.17 Glycolysis II, Gluconeogenesis I

  44. Going the other way… • If there’s lots of glucose, [glucagon] falls • At the same time, [F-1-P] goes up because glycolysis has more fuel • Therefore more F-2,6-P is made: • F-1-P is a substrate for the PFK-2 reaction • F-1-P is an inhibitor of the phosphatase activity of the bifunctional PFK-2 enzyme • So that activates PFK-1 • So more of the glucose gets processed into pyruvate Glycolysis II, Gluconeogenesis I

  45. Pyruvate kinase regulation • 4 isozymes in mammals • Liver, kidney, erythrocyte versions:activated by F-1,6-bisP, inhibited by ATP • Liver, intestinal version: phosphorylated by protein kinase A in response to glucagon release (usually with low [glucose]);That inactivates pyruvate kinase • Also regulated via transcriptional control: more pyruvate kinase made when diet contains a lot of carbohydrate Glycolysis II, Gluconeogenesis I

  46. Pasteur effect • Glycolysis slows down in the presence of oxygen in yeast • This makes sense, since oxygen means we can use oxidative phosphorylation, which is much more efficient than anaerobic glycolysis • How does it work? Several mechanisms, incl.: • O2 inactivates PFK-1 via decreases in AMP Glycolysis II, Gluconeogenesis I

  47. Fructose • Transported with GLUT5 • Ordinarily phosphorylated to F-1-Pby ATP-dependent fructokinase • F-1-P cleaved to DHAP and glyceraldehyde by aldolase • Glyceraldehyde is 3-phosphorylated by ATP-dependent triose kinase • DHAP, Glyc-3-P then enter glycolysis as usual fructose Glycolysis II, Gluconeogenesis I

  48. Fructose-metabolizing enzymes • Fructokinase • F-1P aldolase(now considered a subset of ordinary F-1,6-bisP aldolase) • Triose kinase (no structures yet!) Fructokinase PDB 2hlzhuman136 kDa tetramer Glycolysis II, Gluconeogenesis I

  49. Fructose:Energetics and regulation • Net energetics equivalent to glucose: net 2 ATPs + 2 NADHs per input C6 sugar • This pathway bypasses PFK-1 so it’s less carefully regulated • Therefore diets high in fructose can give rise to fatty livers after accumulations of pyruvate Glycolysis II, Gluconeogenesis I

  50. Sucrose’s fate • In humans there are secreted invertases that split sucrose into fructose + glucose • Plants have sucrose transporters and phosphorylases Glycolysis II, Gluconeogenesis I

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