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TCA Cycle II; Pentose Phosphate Pathway

Explore the details of TCA Cycle II and the Pentose Phosphate Pathway with a focus on key reactions, enzymes, and energy production. This introductory biochemistry overview covers the significance and control of these metabolic pathways. Get insights into glucose metabolism and ATP production in aerobic catabolism. Dive into the mechanisms of oxidative phosphorylation and electron transport chain. Discover the intricate balance of energy generation in cellular respiration.

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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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