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Glycogen metabolism; TCA Cycle I

Glycogen metabolism; TCA Cycle I. 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

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Glycogen metabolism; TCA Cycle I

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  1. Glycogen metabolism; TCA Cycle I 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 • G3PDH:will add a structure later 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. 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

  17. 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

  18. Glycogen structure Carbo metabolism, cont'd

  19. 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

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

  21. 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

  22. 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

  23. 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

  24. 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

  25. When are futile cycles used? • Homeothermic organisms (mammals and birds) deliberately waste energy in the form of oxidation of reduced compounds, decoupling oxidation from ADP phosphorylation, to produce heat and maintain body temperatures • Typical body temp: 34-40 C (37 in humans) • Why that range? • Lower: reactions would be slow • Higher: too much heat liberated into environment • Higher: enzymes might start denaturing Carbo metabolism, cont'd

  26. iClicker question #2 The oxidative steps in the pentose phosphate pathway are • (a) at the beginning of the pathway • (b) responsible for producing NADPH • (c) identical to reactions in the Entner-Doudoroff pathway • (d) all of the above • (e) none of the above Carbo metabolism, cont'd

  27. iClicker question #3 Glycogen phosphorylase is • (a) activated by being phosphorylated • (b) inactivated by being phosphorylated • (c) not subject to phosphorylation • (d) tetrameric • (e) none of the above Carbo metabolism, cont'd

  28. Pyruvate to acetyl CoA • We’ve already told you that pyruvate has at least 4 fates • The one we’ll concentrate on now is the decarboxylation and dehydrogenation of pyruvate to acetyl CoA • Acetyl CoA is a building block employed in the TCA cycle and in lipid synthesis • It’s also an intermediate in amino acid catabolism Carbo metabolism, cont'd

  29. Acetyl CoA:a reminder Carbo metabolism, cont'd

  30. 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 Carbo metabolism, cont'd

  31. Sources of acetyl CoA glycogen glucose lactatepyruvate fatty acids amino acidsAcetyl-CoA TCA Carbo metabolism, cont'd

  32. Pyruvate Dehydrogenase • 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 Carbo metabolism, cont'd

  33. The pyruvate dehydrogenase reaction is irreversible • Significance:acetyl CoA cannot be converted back to pyruvate • Therefore fat can’t be converted to carbohydrate • … except in organisms with a glyoxalate shunt Carbo metabolism, cont'd

  34. 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. • lack of thiamine = beriberi • dihydrolipoyl transacetylase (=E2)has coenzymes lipoate and CoA Carbo metabolism, cont'd

  35. Three-component enzyme pyruvate Coenzymes:FAD and NAD+ E1 TPP E2 FAD lipoate E3 CoASH NAD Carbo metabolism, cont'd

  36. TPP and lipoate Carbo metabolism, cont'd

  37. 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 Carbo metabolism, cont'd

  38. 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 Carbo metabolism, cont'd

  39. Transfer to lipoamide on E2 HETPP + lipoamide-E2 TPP + acetyl-dihydrolipoamide Carbo metabolism, cont'd

  40. E2 component • Dihydrolipoyl transacetylase:contains lipoate and CoA Core domain of E2 PDB 1EAA, EC 2.3.1.12 24-mer of 27kDa monomers Azetobacter vinelandii Cubic 432 symmetry Carbo metabolism, cont'd

  41. 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. Carbo metabolism, cont'd

  42. E3 component • Dihydrolipoamide dehydrogenase • Regenerates FADH2 • In next sub-step, NAD is reduced and FAD is regenerated Human E3PDB 1ZMD420 kDa, 2.1 ÅheterooctamerEC 1.8.1.4 Carbo metabolism, cont'd

  43. 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 Carbo metabolism, cont'd

  44. 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 • E1 activated by dephosphorylation with a phosphatase Carbo metabolism, cont'd

  45. Control of E1 phosporylation state • Kinase (inactivates E1) • Activated by acetyl CoA, NADH • Inhibited by pyruvate, ADP • Phosphatase (activates E1) • Activated indirectly by insulin • Inhibited by acetyl CoA, NADH (I think) Carbo metabolism, cont'd

  46. Defining the Krebs cycle 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 Carbo metabolism, cont'd

  47. Energetics of the cycle • One high energy compound is produced directly for each 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. Carbo metabolism, cont'd

  48. Significance • TCA cycle is central to all respiratory oxidation, 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 Carbo metabolism, cont'd

  49. The TCA cycle • Chart courtesy U.Guelph Carbo metabolism, cont'd

  50. Overall TCA cycle Reaction acetyl-CoA + 3NAD+ + FAD + GDP + Pi + 2H2O 2CO2 + 3NADH + FADH2 + GTP + 2 H+ + CoA • Both carbons oxidized: • One GTP • Three NADH • One FADH2 Carbo metabolism, cont'd

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