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Carbohydrate metabolism II

Carbohydrate metabolism II. Andy Howard Introductory Biochemistry 27 October 2010. Precursors for gluconeogenesis Regulation of gluconeogenesis Pentose phosphate pathway Glyoxylate Shunt Glycogen. Pyruvate to Acetyl CoA Pyruvate Dehydrogenase Control TCA Cycle Acetyl CoA to citrate

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Carbohydrate metabolism II

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  1. Carbohydrate metabolism II Andy HowardIntroductory Biochemistry27 October 2010 Carbo metabolism, cont'd

  2. Precursors for gluconeogenesis Regulation of gluconeogenesis Pentose phosphate pathway Glyoxylate Shunt Glycogen Pyruvate to Acetyl CoA Pyruvate Dehydrogenase Control TCA Cycle Acetyl CoA to citrate Citrate to isocitrate Isocitrate to -ketoglutarate What we’ll discuss Carbo metabolism, cont'd

  3. Substrates for gluconeogenesis: Pyruvate Lactate TCA cycle intermediates Most amino acids Not substrates for gluconeogenesis Acetyl-CoA(at least, not in mammals) Fatty acids Lysine Leucine Substrates and non-substrates Carbo metabolism, cont'd

  4. Lactate as gluconeogenesis precursor • Lactate to pyruvate via lactate dehydrogenase • Comes from glycolysis in muscle • Cori cycle: • lactate in muscle travels to liver • serves as gluconeogenesis substrate • glucose goes back to the muscle • Energy shortfall derived from fatty acid oxidation in the liver Carbo metabolism, cont'd

  5. Amino acids as sources for gluconeogenesis • Most amino acids (“glucogenic”) wind up metabolized to either pyruvate or TCA cycle intermediates • Alanine, serine, … to pyruvate • Aspartate  fumarate  malate  OAA • These can make it back to glucose • A few (“ketogenic”) aa’s do not • We’ll look at this in detail in ch. 17 Carbo metabolism, cont'd

  6. Triacylglycerol as a source • Triacylglycerols  glycerol + FAs • Fatty acids  acetyl CoA + NADH • Glyoxalate cycle (see below) can convert acetyl CoA to glucose; not in mammals • Glycerol: phosphorylated to glycerol-3-P • Glycerol-3-P to DHAP in liver: • Mitochondrial glycerol-3-P dehydrogenase in inner mitochondrial membrane; produces QH2 • Cytosolic G3PDH produces NADH Carbo metabolism, cont'd

  7. Enzymes in glycerol metabolism Glycerol kinase 3H3N • Glycerol kinase • ATP-dependent • Phosphorylation of H232 increases activity • Glycerol-3P DH:may be a drug target for Trypanosomes Glycerol-3-P DHPDB 1EVYLeishmania1.75Å; 78 kDa dimer Carbo metabolism, cont'd

  8. Propionate • Ruminants (cattle, sheep, etc.) have microorganisms in their rumen that break down cellulose and other plant material • Lots of propionate and lactate produced • Propionate  propionyl CoA  succinyl CoA, which is a TCA cycle intermediate Carbo metabolism, cont'd

  9. Acetate • Some species can convert acetate to acetyl CoA • If they have a glyoxalate cycle they can then make that into OAA and get net glucose production • In bacteria that can make acetate from CO2, this allows synthesis of glucose from inorganic source Carbo metabolism, cont'd

  10. Regulation of gluconeogenesis • It’s reciprocally regulated with respect to glycolysis • Primary short-term control points: • Pyruvate  OAA  PEP:Pyr carboxylase accelerated by acetyl CoA • Fructose 1,6-bisP  Fructose-6-P:Inhibited by AMP, F-2,6-bisP • Glucagon activity: see last lecture Carbo metabolism, cont'd

  11. Regulation by [substrate] • Amino acids (ala, others) and lactate are primary substrates for gluconeogenesis • These pathways aren’t saturated: • increasing [aa] in blood means more glucose gets made • Increased [lactate] makes more glucose • Since lactate comes from muscle, liver activity is influenced by other locations Carbo metabolism, cont'd

  12. Routes to oxaloacetate Carbo metabolism, cont'd

  13. Found in bacteria,protists, fungi, and plants Acetyl CoA to OAA via glyoxalate cycle Carbo metabolism, cont'd

  14. Glyoxalate cycle and TCA cycle Carbo metabolism, cont'd

  15. iClicker quiz question 1 Which of the following is not an important precursor for gluconeogenesis in mammals? • (a) Alanine • (b) Lactate • (c) Acetate • (d) Glycerol • (e) All for of these work in mammals Carbo metabolism, cont'd

  16. Pentose Phosphate Pathway • Recall that NAD+/NADH is primarily involved in catabolic pathways • NADP+/NADPH primarily biosynthetic • PPP supplies NADPH for reductive biosynthetic reactions • PPP also known as hexose monophosphate shunt or phosphogluconate pathway • Operates mostly in cytosol of liver & adipose cells: needed there for fatty acid synthesis Carbo metabolism, cont'd

  17. Overview of PPP • Begins with oxidation of glucose 6-phosphate to 6-phosphogluconate (C6-P  C6-P) • That gets decarboxylated toribulose-5-phosphate (C6-P  C5-P) • RuB-5-P isomerized to ribose-5-P & epimerized to xylulose-5-P (2C5-P  C5-P + C5-P) • These recombine forming sedoheptulose-7-P and glyceraldehyde-3-P (C5-P+C5-P  C7-P + C3-P) • Those are reorganized to erythrose-4-P + fructose-6-P (C7-P+C3-P  C4-P + C6-P) • Erythrose-4-P + another xylulose-5-P reorganized to another fructose-6-P + another glyceraldehyde-3-P (C4-P+C5-P  C6-P + C3-P) Carbo metabolism, cont'd

  18. How the products are used • Oxidative reactions at the beginning • NADPH produced in oxidation of gluc-6-P and in decarboxylation of 6-phosphogluconate • NADPH used in fatty acid synthesis, other anabolic reactions • The rest are nonoxidative; their role is to provide metabolites for other paths • Glyceraldehyde-3-P and fructose-6-P can re-enter glycolysis or gluconeogenesis • Ribose-5-P used in nucleic acid biosynthesis Carbo metabolism, cont'd

  19. First PPP reaction: G6PDH • Glucose-6-phosphatederived from glycogenvia Gluc-1-P) orgluconeogenesis • Reaction:Gluc-6-P + NADP + H2O 6-P-gluconolactone + NADPH + H+ • Irreversible, highly regulated reaction 6-P-gluconolactone Carbo metabolism, cont'd

  20. Glucose 6-phosphate dehydrogenase • Inhibited by NADPH, fatty acyl esters of CoA (downstream reaction products) • Regulated by cytosolic [NADP+]/[NADPH] ~ 0.015; contrast with[NAD+]/[NADH] ~ 725 Human G6PDHPDB 2BH9, 2.5ÅEC 1.1.1.49116 kDa dimermonomer shown Carbo metabolism, cont'd

  21. Medical significance (Box 12.2) • 2 isozymes of this enzyme in humans • G6PDH - gene on X chromosome;mostly found in erythrocytes • H6PDH (less substrate-specific):found in other tissues • Many (4%?) have G6PDH abnormalities leading to hemolytic anemia: NADPH deficiencies lead to rapid breakdown of RBCs • Mutations survive in gene pool because of heightened resistance to malaria among victims Carbo metabolism, cont'd

  22. Equilibrium for sugar acid • As we discussed, 6-phosphogluconolactone is somewhat unstable • Ring spontaneously opens to form 6-phosphogluconate • … but the enzyme gluconolactonase helps that along Carbo metabolism, cont'd

  23. Gluconolactonase • Ca2+ or Zn2+ forms exist • 7 Ca2+ per dimer: one in interface, 3 in each monomer • Extensive interface between monomers • Overlap seen at right Xanthamonas gluconolactonase 66 kDa dimer EC 3.1.1.17, 1.6ÅPDB 3DR2 Carbo metabolism, cont'd

  24. Path to ribulose-6-P • 2nd NADPH-yielding step: 2 sub-steps • 6-P-gluconate reacts with NADP+ to yieldNADPH + H+ + 3-keto-6-P-gluconate • That then gets decarboxylated to yield D-ribulose-6-P • -keto-acids like this are strongly subject to decarboxylation 3-keto-6-P-gluconate Carbo metabolism, cont'd

  25. 6-phosphogluconate dehydrogenase • Typical Rossmann fold NAD(P) binding domain • Helical domain at subunit interface • Catalyzes both parts of reaction; hydride transfer to NADP precedes decarboxylation Human 6-P-gluconatedehydrogenasePDB 2JKV EC 1.1.1.44, 2.53Å340 kDa hexamerdimer shown Carbo metabolism, cont'd

  26. Nonoxidative steps • All the remaining PPP steps are near-equilibrium, reversible, non-oxidative steps • Role (as discussed above) is interconversion of sugar phosphates • Supplies ribose-5-P (e.g. to nucleotide synthesis) and glycolytic intermediates Carbo metabolism, cont'd

  27. Fates of ribulose 5-phosphate • Either the carbonyl moves from 2 to 1 (isomerase) or the chirality changes at C3 (epimerase) • Epimerase leads to xylulose 5-phosphate • Isomerase leads to ribose 5-phosphate • Both reactions proceed via enediolate intermediates (2,3) or (1,2) Carbo metabolism, cont'd

  28. Epimerization • Ribulose-5-P 2,3-enediolate  xylulose 5-P • Allows for switching chirality at C3 • Enzymatically controlled 2,3-enediolate ofribulose-5-P Carbo metabolism, cont'd

  29. Ribulose 5-phosphate epimerase • TIM-barrel structure • One barrel per monomer • Example of the fact that TIM barrels are promiscuous in their applicability SynechocystisRibulose 5-P epimerase, PDB 1TQJEC 5.1.3.1, 1.6Å150 kDa hexamer Carbo metabolism, cont'd

  30. Isomerization • Rub-5-P 1,2-enediolate  Ribose-5-P • Swaps carbonyl from C-2 to C-1 • Enzymatically controlled 1,2-enediolate ofribulose-5-P Carbo metabolism, cont'd

  31. E.coli RPIAPDB 1O8B 46.9kDa dimer EC 5.3.1.6, 1.3Å Ribose-5-phosphate isomerase(RPIA) • Each monomer is a 2-layer sandwich • Highly conserved protein Carbo metabolism, cont'd

  32. Medical significances of RPIA • Deficiencies in human RPIA lead to leukoencephalopathy because of accumulations of pentoses and pentose phosphates • Plasmodium relies heavily on PPP, partly because they need to use NADPH to break down heme; so P.falciparum’s RPIA is seen as target in anti-malarial drug design Carbo metabolism, cont'd

  33. Fate of xylulose 5-P and ribose 5-P • Ribose 5-P: precursor to nucleotides • Xylulose 5-P: used in a later step … • These 2 together are substrates for a transketolase reaction (5+57+3): • Products are sedoeptulose 7-phosphate and glyceraldehyde 3-phosphate Sedo-heptulose 7-P Carbo metabolism, cont'd

  34. Transketolase • TPP-dependent enzyme • Transfers 2-carbon glycoaldehyde group from a ketose to an aldose • Effect is to shorten the ketose by 2 C’s while converting it to an aldose; and • … to lengthen the aldose by 2 P’s while converting it to a ketose. • In this case C5+C5  C7 + C3, but that is dependent on substrates, obviously Campylobacter transketolasePDB 3L84EC 2.2.1.1, 1.3Å140kDa dimer; monomer shown Carbo metabolism, cont'd

  35. Mechanism of transketolase • See transketolase Wikipedia article • Roughly symmetric mechanism(like serine protease) • Glu418, His263 involved on both sides • Both the thiazolium ring and the thymine ring of TPP are directly involved Carbo metabolism, cont'd

  36. Transaldolase reaction • Sedoheptulose 7-P not terribly useful • Transaldolase converts C7+C3 to C4+C6 • Sedoheptulose 7-P + glyceraldehyde 3-P  erythrose 4-P + fructose 6-P • Effectively we’re moving 3 carbons from the ketose onto the aldose, converting the C7 ketose to a C4 aldose and converting the C3 aldose to a C6 ketose Carbo metabolism, cont'd

  37. Transaldolase • Aldolase-style TIM barrel • This bacterial version is a monomer • Human enzyme is a dimer with a specific dimerization domain holding the dimer together Neisseria transaldolasePDB 3CLMEC 2.2.1.2, 1.14Å38.6 kDa monomer Carbo metabolism, cont'd

  38. Transaldolase mechanism • Like the aldolase in glycolysis • Schiff-base formed between sedoheptulose 7-P and a lysine to form an eneamine • That releases the 4-carbon aldose • Glyceraldehyde 3-P then abstracts the 3-carbon entity to form into a 6-carbon ketose Carbo metabolism, cont'd

  39. Second transketolase • Another xylulose-5-P enters: reacts with erythrose-4-P produced in the transaldolase reaction (C5+C4 C3+C6) • Same enzyme as two steps ago • Products are another fructose 6-P and a glyceraldehyde 3-P • So now we’re done with this system! Carbo metabolism, cont'd

  40. How is glucose 6-P used? • Depends on needs for ribose 5-P, NADPH, and ATP • Certain reactions within this system and in glycolysis are re-used in ways that produce whatever is needed most • So the number of glucose 6-P molecules used to produce the various products will depend on what is in short supply Carbo metabolism, cont'd

  41. Relationship between PPP and other pathways • First two reactions are shared with the Entner-Doudoroff pathway, which you’ve already seen • Several reactions in common with the Calvin cycle (dark reactions of photosynthesis), which we’ll meet soon • Substrates derived from glycolysis and glycogen degradation • Products used in anabolic reactions and in making nucleotides Carbo metabolism, cont'd

  42. Glycogen • Remember that this is the primary middle-term storage molecule for sugars in animals • In mammals, most glycogen storage occurs in the liver • Glycogen is a homopolymer of glucopyranose units, typically 50000 units(~ 810 kDa) • Most links are -1,4 • About 10% are -1,6 (crosslinks) Carbo metabolism, cont'd

  43. Glycogen (review) • Principal storage form of glucose in human liver; some in muscle • Branched (a-14 + a few a-16) • More branches (~10%) than in amylopectin • Larger than starch: 50000 glucose • One reducing end, many nonreducing ends • Broken down to G-1-P units • Built up fromG-6-P  G-1-P  UDP-Glucose units Carbo metabolism, cont'd

  44. Glycogen structure Carbo metabolism, cont'd

  45. Glycogen breakdown • Start with glycogen; phosphorylate the nonreducing, terminal glucose group(glycogen)n + Pi(glycogen)n-1 + glucose-1-phosphate • Enzyme involved is glycogen phosphorylase • glucose 1-phosphate can be isomerized to glucose 6-phosphate with the help of phosphoglucomutase Carbo metabolism, cont'd

  46. Rate of glycogen replenishmentafter exhaustive exercise Carbo metabolism, cont'd

  47. Rabbit muscle Glycogen phosphorylasePDB 2GJ4, 1.6ÅEC 2.4.1.1 193 kDa dimermonomer shown Control of glycogen breakdown • Glycogen phosphorylase exists in a relatively inactive, unphosphorylated formcalled phosphorylase b • Phosphorylated to form phosphorylase a, which is much more active • ATP, Glucose-6-P inhibit phosphorylase • AMP activates it(makes sense!) Carbo metabolism, cont'd

  48. Glycogen synthesis • Glycogen synthase (fig. 12.11) catalyzes addition of UDP-glucose units to glycogen: • UDP-glucose + (glycogen)n UDP + (glycogen)n+1 • Initiation and branch formation involve different enzymes E.coli (ADP-glucose) glycogen synthase PDB 2QZS, 2.2Å EC 2.4.1.21 55 kDa monomer Carbo metabolism, cont'd

  49. Reciprocal control • Conditions that activate glycogen synthesis are precisely the conditions that deactivate glycogen breakdown • Conditions that activate glycogen breakdown are precisely the conditions that deactivate glycogen synthesis • Phosphorylated glycogen synthase is the inactive form; phosphorylated glycogen phosphorylase is the active form (scheme 12.14) • Result: avoidance of futile cycles(breakdown accompanying buildup) Carbo metabolism, cont'd

  50. Why is that useful? • Futile cycles waste energy • Energy loss usually takes the form of heat • Generally organisms avoid that! • Exception: homeothermic organisms • Glycogen futile cycles are avoided Carbo metabolism, cont'd

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