1 / 94

Pentose Phosphate Pathway & TCA Cycle

Pentose Phosphate Pathway & TCA Cycle. Andy Howard Biochemistry Lectures, Spring 2019 4 April 2019. PPP & TCA cycle matter. Pentose Phosphate Pathway is a source of reducing equivalents and specific monosaccharides

jtommie
Download Presentation

Pentose Phosphate Pathway & TCA Cycle

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Pentose Phosphate Pathway & TCA Cycle Andy HowardBiochemistry Lectures, Spring 20194 April 2019

  2. PPP & TCA cycle matter • Pentose Phosphate Pathway is a source of reducing equivalents and specific monosaccharides • TCA cycle is the primary route through which sugars provide ATP and NAD for cellular energy PPP & TCA Cycle

  3. Pentose Phosphate pathway Non-oxidative steps Significance Pyruvate Overview PDC & control TCA Cycle Steps through Succinyl CoA Steps to oxaloacetate Control Anapleurotic reactions Energetics Evolution What we’ll discuss PPP & TCA Cycle

  4. Nonoxidative steps in PPP • Remaining PPP steps are near-equilibrium, reversible, non-oxidative steps • Role is interconversion of sugar phosphates • Supplies ribose-5-P (e.g. to nucleotide synthesis) and glycolytic intermediates Ribulose 5-phosphate PPP & TCA Cycle

  5. Overview of PPP’s nonoxidative steps • 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 andfructose-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) PPP & TCA Cycle

  6. 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) PPP & TCA Cycle

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

  8. Ribulose 5-phosphate epimerase • TIM-barrel structure • One barrel per monomer • Example of the fact that TIM barrels are promiscuous in their applicability Neisseria Ribulose 5-P epimerase, EC 5.1.3.1154 kDa hexamerPDB 5UMF, 1.4Å PPP & TCA Cycle

  9. Isomerization • Ribulose-5-P 1,2-enediolate  Ribose-5-P • Swaps carbonyl from C-2 to C-1 • Enzymatically controlled 1,2-enediolate ofribulose-5-P PPP & TCA Cycle

  10. E.coli RPIA46.9kDa dimerEC 5.3.1.6PDB 1O8B, 1.3Å Ribose-5-phosphate isomerase (RPIA) • Each monomer is a2-layer sandwich • Highly conserved protein PPP & TCA Cycle

  11. Medical significances • 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 Plasmodium RPIAEC 5.3.1.656 kDa dimerPDB 2F8M, 2.1Å PPP & TCA Cycle

  12. 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 sedoheptulose 7-phosphate and glyceraldehyde 3-phosphate sedo-heptulose 7-P PPP & TCA Cycle

  13. 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 C’s while converting it to a ketose. Human transketolaseEC 2.2.1.1142kDa dimer; monomer shownPDB 4KXV, 0.97Å PPP & TCA Cycle

  14. Transketolase in this context In this case C5+C5  C7 + C3, but that is dependent on substrates, obviously In a later step it’ll be different; but it’s always adding 2 to the aldose, subtracting 2 from the ketose, and then switching their identities PPP & TCA Cycle

  15. 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 PPP & TCA Cycle

  16. Transaldolase significance • 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 PPP & TCA Cycle

  17. 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 transaldolaseEC 2.2.1.238.6 kDa monomerPDB 3CLM, 1.14Å PPP & TCA Cycle

  18. 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 PPP & TCA Cycle

  19. Second transketolase step • 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! PPP & TCA Cycle

  20. 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 PPP & TCA Cycle

  21. Relationship between PPP and other pathways • First two reactions are shared with the Entner-Doudoroff pathway • Several reactions in common with the Calvin cycle (dark reactions of photosynthesis), which we’ll meet soon PPP & TCA Cycle

  22. iClicker question #1 1. Which enzyme is used in two separate nonoxidative steps in the PPP? • (a) G6PDH • (b) citrate synthase • (c) transaldolase • (d) transketolase • (e) none of the above PPP & TCA Cycle

  23. Pyruvate to acetyl CoA • We’ve already told you that pyruvate has >= 4 fates • We’ll now concentrate on the decarboxylation and dehydrogenation of pyruvate to acetyl CoA • Acetyl CoA is a building block employed in the TCA cycle and in lipid synthesis • Acetyl CoA also an intermediate in amino acid catabolism PPP & TCA Cycle

  24. Coenzyme A:a reminder PPP & TCA Cycle

  25. 3 stagesof cellular respiration 1. Acetyl CoA production—fromglucose, fatty acids, amino acids 2. Acetyl CoA oxidation via theTCA Cycle:yields reduced electron carriers 3. Electron transport and oxidative phosphorylation: • oxidation of these electron carriers • production of ATP PPP & TCA Cycle

  26. Sources of acetyl CoA glycogen glucose lactatepyruvate fatty acids amino acidsAcetyl-CoA TCA PPP & TCA Cycle

  27. Pyruvate Dehydrogenase(CF&M §19.3) • Converts pyruvate to acetyl CoA • Setup enzyme for the TCA cycle • Located in mitochondrial matrix • Net reaction:pyruvate + NAD+ + HS-CoA acetyl CoA + NADH + CO2 PPP & TCA Cycle

  28. The pyruvate dehydrogenase reaction is irreversible • Significance: acetyl CoA cannot be converted back to pyruvate • Therefore even-chain fatty acids can’t be converted to carbohydrate • … except in organisms with a glyoxalate pathway PPP & TCA Cycle

  29. Pyruvate Dehydrogenase:Enzyme Properties • 2.5 x 106 Da complex:multiple copies of 3 enzymes, E1, E2, E3 • pyruvate decarboxylase (=E1) uses the coenzyme thiamine pyrophosphate (TPP) • TPP is coenzyme for alldecarboxylations of -keto acids. • dihydrolipoyl transacetylase (=E2)has coenzymes lipoate and CoASH PPP & TCA Cycle

  30. Three-component enzyme system pyruvate Coenzymes:FAD and NAD+ E1 TPP E2 FAD lipoate E3 CoASH NAD PPP & TCA Cycle

  31. TPP and lipoate PPP & TCA Cycle

  32. Partial reactions of PDH • 1st reaction:catalyzed by pyruvate decarboxylase (E1)proceeds via ylid intermediate: • Pyruvate + thiamine pyrophosphate + H+hydroxyethylthiamine pyrophosphate carbanion + CO2 + CH3COCOO- + CO2 PPP & TCA Cycle

  33. E1 component • Responsible for TPP-dependent decarboxylase portion of mechanism • 2 active center loops become ordered only after they bind a substrate that can form a stable intermediate with TPP E1 of E.coli pyruvate dehydrogenase PDB 2QTC, 1.8Å EC 1.2.4.1 200.4 kDa dimer PPP & TCA Cycle

  34. Transfer to lipoamide on E2 HETPP + lipoamide-E2 TPP + acetyl-dihydrolipoamide PPP & TCA Cycle

  35. E2 component • Dihydrolipoyltransacetylase:contains lipoate and CoA Azetobacter vinelandiiCore domain of E224-mer of 27kDa monomersCubic 432 symmetry EC 2.3.1.12, PDB 1EAA, 2.6Å PPP & TCA Cycle

  36. Steps involving E2 and E3 1. E2 then transfers acetyl to CoA; acetyl-CoA leaves. 2. E3 uses its bound coenzyme FAD to oxidize lipoamide back to disulfide and generating FADH2. 3. FAD is recovered from FADH2via reducing NAD to NADH;An NADH is generated. PPP & TCA Cycle

  37. E3 component • Dihydrolipoamide dehydrogenase • Regenerates FADH2 • In next sub-step, NAD is reduced and FAD is regenerated Pseudomonas E3210 kDa homotetramerEC 1.8.1.4; PDB 5U8U, 1.4Å PPP & TCA Cycle

  38. Regulation of Mammalian Pyruvate Dehydrogenase Irreversible reaction must be tightly controlled-- three ways 1. Allosteric Inhibition • inhibited by products: acetyl-CoA and NADH • inhibited by high ATP 2. Allosteric activation by AMP:Ratio of ATP/AMP important PPP & TCA Cycle

  39. Phosphorylation/dephosphorylation of E1 • You would expect that an important, irreversible reaction like E1 would be subject to allostery or PTM • … and you’d be right • E1 is inactivated by phosphorylation with an ATP-dependent kinase • E1activated by dephosphorylation with a phosphatase PPP & TCA Cycle

  40. Pyruvate Dehydrogenase Kinase • Inactivates E1 • Activated by acetyl CoA, NADH, ATP • Inhibited by pyruvate, ADP, NAD, CoASH • PDK4 overexpressed in insulin-free cells, so potential diabetes target Human PDK Isoform 2EC 2.7.11.291kDa dimer, monomer shownPDB 5J71, 1.65Å PPP & TCA Cycle

  41. Pyruvate Dehydrogenase Phosphatase Bovine PDP1CEC 3.1.3.4353kDa monomerPDB 3N3C, 2.1Å Activates E1 Mn2+-dependent mitochondrial enzyme Activated indirectly by insulin, PEP, AMP Inhibited by acetyl CoA, NADH, ATP PPP & TCA Cycle

  42. Defining the TCA cycle(CF&M 19.1,2,4) 3 roughly equivalent names:tricarboxylic acid cycle; Krebs cycle; citric acid cycle Definition: Acetyl CoA is derived from pyruvate and other metabolites And is oxidized to CO2 in this cycle PPP & TCA Cycle

  43. Energetics of the cycle (CF&M §19.5) • One high energy compound (GTP or ATP) is produced directly for each round of the cycle. • The electrons from the TCA cycle are made available to an electron transport chain in the form of three NADH and one FADH2 and ultimately energy is provided for oxidative phosphorylation. PPP & TCA Cycle

  44. Significance (CF&M §19.1) • TCA cycle is central to all respiratory oxidation in aerobic organisms, oxidizing acetyl-CoA from glucose, lipid and protein catabolism in aerobic respiration to maximize energy gain. • Also supplies some precursors for biosynthesis. • All enzymes are in the mitochondrial matrix or inner mitochondrial membrane PPP & TCA Cycle

  45. The TCA cycle • Chart courtesy U.Guelph PPP & TCA Cycle

  46. Overall TCA cycle Reaction acetyl-CoA + 3NAD+ + FAD + GDP + Pi + 2H2O 2CO2 + 3NADH + FADH2 + GTP + 2 H+ + CoA • Both of the carbons derived from acetyl CoA are oxidized into carbon dioxide, yielding: • One GTP • Three NADH • One FADH2 PPP & TCA Cycle

  47. Nomenclature for dicarboxylic acids • TCA cycle involves a few tricarboxylic acids and a bunch of dicarboxylic acids • Underivatized dicarboxylic acids have trivial names up through at least 10 C’s. • If we get comfortable with the first few, it will help a lot in the days to come. • -OOC–(CH2)n–COO- PPP & TCA Cycle

  48. #C #CH2 Name 2 0 oxalate 3 1 malonate 4 2 succinate 5 3 glutarate 6 4 adipate #C #CH2 Name 7 5 pimelate 8 6 suberate 9 7 azelate 10 8 sebacate Dicarboxylic acids Oh my, such good apple pie, sweet as sugar! PPP & TCA Cycle

  49. 1- Condensing acetyl-CoA with oxaloacetate (oxosuccinate) Enzyme: citrate synthase PPP & TCA Cycle

  50. Citrate Synthase Reaction • committing step in TCA cycle • highly exergonic, driven largely by cleavage of thiol ester bond of acetyl CoA Thermus citrate synthase87 kDa dimermonomer shownEC 2.3.3.1PDB 1IOM, 1.5Å PPP & TCA Cycle

More Related