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TCA Cycle II; Pentose Phosphate Pathway

TCA Cycle II; Pentose Phosphate Pathway. Andy Howard Introductory Biochemistry 24 March 2010 Based in part on a U.Florida lecture. TCA cycle  -ketoglutarate to succinate Succinate to oxaloacetate Control Anapleurotic reactions. Pentose phosphate pathway Overview Significance Specifics

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TCA Cycle II; Pentose Phosphate Pathway

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  1. TCA Cycle II;Pentose Phosphate Pathway Andy HowardIntroductory Biochemistry24 March 2010 Based in part on a U.Florida lecture TCA Cycle II; PPP

  2. TCA cycle -ketoglutarate to succinate Succinate to oxaloacetate Control Anapleurotic reactions Pentose phosphate pathway Overview Significance Specifics Control What we’ll discuss TCA Cycle II; PPP

  3. The TCA cycle • Chart courtesy U.Guelph TCA Cycle II; PPP

  4. Overall TCA cycle Reaction(review) acetyl-CoA + 3NAD+ + FAD + GDP + Pi + 2H2O 2CO2 + 3NADH + FADH2 + GTP + 2 H+ + CoA • Both carbons oxidized: • One GTP • Three NADH • One FADH2 TCA Cycle II; PPP

  5. 4- Oxidation of -ketoglutarate to succinyl-CoA and CO2 • Enzyme = -ketoglutaratedehydrogenase complex • -ketoglutarate + HS-CoA + NAD+ NADH + CO2 + succinyl CoA  TCA Cycle II; PPP

  6. Characteristics of -ketoglutarate dehydrogenase reaction • Second oxidation in TCA cycle;another NADH is produced • Loss of second of two CO2 • Highly exergonic;G°’ = -33.5 kJ mol-1 • Reaction similar to pyruvate to acetyl-CoA • Enzyme is similar to pyruvate dehydrogenase complex (E3 same) • The substrate is pyruvate + carboxymethylene E1 of KGADH PDB 2JGD205 kDa dimer E.coli; 2.6ÅEC 1.2.4.2 TCA Cycle II; PPP

  7. E2 component of KGDH • Catalytic domain has cubic (432) quaternary structure:24 equivalent subunits • We saw this with pyruvate dehydrogenase as well TCA Cycle II; PPP

  8. 5: Succinyl CoA to succinate • Enzyme: succinyl CoA synthetase • Succinyl CoA + Pi + GDP Succinate + HS-CoA + GTP • substrate level phosphorylation • GTP is equivalent to ATP;GTP ATP via nucleoside diphosphate kinase • A few organisms haveATP-dependent versions TCA Cycle II; PPP

  9. Characteristics of the succinyl CoA synthetase reaction • succinyl phosphate is intermediate. • overall near equilibrium;G°’ = -2.9 kJ mol-1 • succinyl CoA has a strong negative free energy for hydrolysis of its thioester; used to synthesize a GTP. PDB 1EUD, 2.1Å76 kDa heterodimer Pig heart, EC 6.2.1.4 TCA Cycle II; PPP

  10. 6- Oxidation of succinate to fumarate • Enzyme: succinate dehydrogenase-- a flavoprotein bound to the innermembrane of the mitochondrionSuccinate + FAD  fumarate + FADH2 • a dehydrogenation;note trans double bond • Preview:the subsequent reoxidation of FADH2 will yield 1.5 ATPs while the reoxidation of NADH will yield 2.5 ATPs. TCA Cycle II; PPP

  11. Succinate Dehydrogenase Reaction • 3rd oxidation of TCA cycle: • FAD in flavoprotein reduced to FADH2 • a flavin-dependent oxidation • electrons are captured herefor electron transport chain • We’ll revisit this in the electron-transport chapter, where we’ll label it as “respiratory complex II” 2H88: 245 kDa, 1.8Å, EC 1.3.5.1 Dimer of heterotetramers: chicken TCA Cycle II; PPP

  12. 7: Hydration of fumarate Thermus fumarase, 1.8Å PDB 1VDKEC 4.2.1.2106 kDa tetramer • Enzyme: fumarase • Fumarate + H2O L-malate (chiral!) TCA Cycle II; PPP

  13. 8- Oxidation of malate to oxaloacetate • It’s a cycle! • Enzyme: malate dehydrogenase • L-malate + NAD+oxaloacetate + NADH + H+ • recall same reaction in gluconeogenesis • fourth oxidation; another pair of electrons is made available in NADH. TCA Cycle II; PPP

  14. Characteristics of MDH reaction • formation of oxaloacetate close to equilibrium • reaction driven by exergonic synthesis of citrate • oxaloacetate concentration is thereby kept low PDB 1Y7T, 1.65Å 72kDa dimer, EC 1.1.1.37Thermus thermophilus TCA Cycle II; PPP

  15. Summary of TCA cycle • First Half -- introduction of two carbon atoms and their loss,yielding 2 NADH and a GTP (= ATP) • Second Half-- partial oxidation of succinate to oxaloacetate. Another NADH is produced as well as a reduced FADH2.Oxaloacetate is regenerated for next cycle. TCA Cycle II; PPP

  16. Discussion question The two carbon atoms that are removed in each cycle are different from the ones put onto oxaloacetate. Exercise question: When are these carbons released? TCA Cycle II; PPP

  17. iClicker question:When are they released? • (a) Into NAD and FAD • (b) Into ADP • (c) By decarboxylation at the dehydrogenase steps • (d) None of the above TCA Cycle II; PPP

  18. Overall TCA cycle reaction • acetyl-CoA + 3NAD+ + FAD + GDP +Pi + 2H2O 2CO2 + 3NADH + FADH2+ GTP + 2H+ + CoA • one high energy compound made • four pairs of electrons are made • available to the respiratory chain and oxidative phosphorylation. • These are used to generate most of the ATP needed. TCA Cycle II; PPP

  19. Glucose to ATP • What is maximum yield of high energy ATP in the aerobic catabolism of glucose? • Glycolysis:glucose  2pyruvate + 2NADH+2ATP 7 ATPs • Pyruvate Dehydrogenase:2pyruvate  2acetyl CoA + 2NADH 5 ATPs • TCA cycle:acetyl CoA  2CO2+3NADH+FADH2+GTP 2x10ATPs • OVERALL yield from glucose 32 ATPs TCA Cycle II; PPP

  20. Is this really right? • It’s tricky to calculate how many ATP molecules can be produced from oxidation of a single molecule of NADH or FADH2. • These 1.5 and 2.5 values are the current consensus, but higher (2,3) values are sometimes found in textbooks and notes, particularly older ones. • With 2 and 3 ATPs per reduced cofactor, you end up with 37 ATP per glucose, not 32 TCA Cycle II; PPP

  21. Energy relationships • G°’ for oxidation of glucose to CO2 is2,840 kJ mol-1 • Much of this energy conserved as ATP:32 ATP X 30.5 kJ mol-1 ATP= 976 kJ mol-1 glucose • This represents 34% conservation of the potential energy available in glucose as ATP. TCA Cycle II; PPP

  22. Regulation of TCA Cycle 1- Pyruvate dehydrogenase • Previously discussed  • inhibited by acetyl-CoA and NADH 2- Citrate synthase[oxaloacetate] is low; that controls rate 3- Isocitrate dehydrogenase • activated allosterically by ADP • inhibited allosterically by NADH 4- -ketoglutarate dehydrogenase • inhibited allosterically by products, namely, succinyl-CoA and NADH (like #1!) TCA Cycle II; PPP

  23. Regulation by NAD/NADH • NOTE: Major regulator is intramitochondrial NAD+/NADH ratio. • Thus, low oxygen level results in decreased ratio and high level an increased ratio. • A measure of oxygen availability TCA Cycle II; PPP

  24. Replacement of intermediates • TCA-cycle intermediates are removed for biosynthesis  • 1- amphibolicreactions =removal of intermediates. • 2- anapleroticreactions =replacing intermediates in the cycle. TCA Cycle II; PPP

  25. TCA cycle-derived compounds a- transaminations:oxaloacetate  Asp removes 4C-ketoglutarate  Glu removes 5Cpyruvate  Ala removes 6C b- fatty acid biosynthesiscitrate  acetyl CoA and oxaloacetateacetyl CoA can build fatty acids c- heme biosynthesissuccinyl CoA + glycine  porphyrins TCA Cycle II; PPP

  26. Anapleurotic reactions a- pyruvate carboxylase • Replaces oxaloacetate • most important, especially in liver and kidney. Note: same reaction in gluconeogenesis TCA Cycle II; PPP

  27. Other anapleurotic reactions Human malic enzymePDB 2AW5253 kDa tetramer EC 1.1.1.40; 2.5Å • b- malic enzyme - • replaces malate • pyruvate + CO2 + NADPH malate + NADP+ • Some versions require NADPH, others NADH • Mn2+ or Mg2+-dependent carboxylase TCA Cycle II; PPP

  28. Anapleurotic reactions based on amino acids c- reversals of transaminations -- restores oxaloacetateor -ketoglutarate with abundant asp or glu using glutamate dehydrogenase glutamate + NAD(P)+-ketoglutarate + NAD(P)H + NH4+ Glutamate dehydrogenase PDB 1GTM; 277 kDadimer of trimers; trimer shown Pyrococcus furiosusEC 1.4.1.3, 2.2Å TCA Cycle II; PPP

  29. The TCA cycle • Chart courtesy U.Guelph TCA Cycle II; PPP

  30. Pentose Phosphate Pathway • Recall that NAD+/NADH is primarily Phosphate 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 TCA Cycle II; PPP

  31. 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) TCA Cycle II; PPP

  32. 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 TCA Cycle II; PPP

  33. 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 TCA Cycle II; PPP

  34. 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 TCA Cycle II; PPP

  35. 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 TCA Cycle II; PPP

  36. 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 TCA Cycle II; PPP

  37. 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 TCA Cycle II; PPP

  38. 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 TCA Cycle II; PPP

  39. 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 TCA Cycle II; PPP

  40. 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 TCA Cycle II; PPP

  41. 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) TCA Cycle II; PPP

  42. Epimerization • Ribulose-5-P 2,3-enediolate  xylulose 5-P • Allows for switching chirality at C3 • Enzymatically controlled 2,3-enediolate ofribulose-5-P TCA Cycle II; PPP

  43. 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 TCA Cycle II; PPP

  44. 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 TCA Cycle II; PPP

  45. E.coli RPIAPDB 1O8B 46.9kDa dimer EC 5.3.1.6, 1.3Å Ribose-5-phosphate isomerase • Each monomer is a 2-layer sandwich • Highly conserved protein TCA Cycle II; PPP

  46. Medical significance 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 TCA Cycle II; PPP

  47. 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 TCA Cycle II; PPP

  48. 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 TCA Cycle II; PPP

  49. 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 TCA Cycle II; PPP

  50. 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 TCA Cycle II; PPP

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