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Energy Metabolism

Energy Metabolism. ATP synthesis Outline the steps of glycolysis Outline the steps of lipolysis Citric acid cycle/Electron transport chain Control processes Explain the contribution of mass action to the rate of ATP synthesis Similarly, allosteric feedback. Phospho-creatine ATP buffer.

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Energy Metabolism

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  1. Energy Metabolism • ATP synthesis • Outline the steps of glycolysis • Outline the steps of lipolysis • Citric acid cycle/Electron transport chain • Control processes • Explain the contribution of mass action to the rate of ATP synthesis • Similarly, allosteric feedback

  2. Phospho-creatine ATP buffer • Creatine Kinase • Unique to striated muscle • Creatine + ATP ADP + phospho-creatine • Creatine • 20-40 mM total creatine • 16-32 mM phospho • ATP ~ 5-10 mM

  3. Glycolysis • Convert Glucose to Pyruvate • Yield 2 ATP + 2 NADH per glucose • Consume 2 ATP to form 2x glyceraldehyde phosphate • Produce 2 ATP + 1 NADH per GAP • Carefully controlled • 12 different enzyme-catalyzed steps • Limited by phosphofructokinase • Limited by substrate availability

  4. Glycolysis: phosphorylation • ATP consuming • Glucose phosphorylation by hexokinase • Fructose phosphorylation by phosphofructokinase • Triose phosphate isomerase

  5. Glycolysis: oxidation • Pyruvate kinase • Transfer Pi to ADP • Driven by oxidative potential of 2’ O • Summary • Start C6H12O6 • End 2xC3H3O3 • Added 0xO • Lost 6xH • Gained 2xNADH, 2xATP GAPDH phosphoglycerate kinase NADH ATP pyruvate kinase

  6. Pyruvate • Lactic Acid • Regenerates NAD+ • Redox neutral • Ethanol • Regenerates NAD+ • Redox neutral • Acetyl-CoA • Pyruvate import to mitocondria • ~15 more ATP per pyruvate S-acetyldihydro-lipoyllysine 2-Hydroxyethyl-Thiamine diphosphate pyruvate Acetyl-CoA

  7. Carbohydrate metabolism depends on transport Major Facilitator Superfamily Monocarboxylate transporter • H+, pyruvate cotransporter Competition between H+ driven transport to mitochondria and NADH/H+ driven conversion to lactate Cytoplasmic NADH is also used to generate mitochondrial FADH2, coupling transport to ETC saturation “glycerol-3P shuttle” Halestrap & Price 1999

  8. Gluconeogenesis • During contraction, inefficient glycolysis wastes glucose • Many glycolytic enzymes are reversible • Special enzymes • Pyruvate carboxylase • Generate 4-C oxaloacetate from 3-C pyruvate • Phosphoenyl pyruvate carboxykinase • Swap carboxyl group for phosphate • Generates 3-C phosphoenolpyruvate from OA • Fructose-1,6-bisphosphatase • Generates fructose-6-phosphate Mitochondrial

  9. Fatty Acid/b-oxidation Cycle • 1x FADH2 • 1x NADH • Acetyl-CoA • 3x NADH+ • 1xFADH2 • Acyl(n)-CoA + NAD+ + FADAcyl(n-2)-CoA + Acetyl-CoA + NADH +FADH2 Carnitine palmitoyltransferase Fatty acid elongation Acyl-CoA Acyl-CoA synthase FAD Acyl-CoA dehydrogenase Acyl-CoA FADH2 acetyl-CoA acyltransferase Acetyl-CoA Didehydroacyl-CoA Acyl-CoA hydrase CoA-SH 3-hydroxyacyl-CoA dehydrogenase Hydroxyacyl-CoA Oxoacyl-CoA NADH NAD+

  10. Reactive oxygen • FADH2 oxidative stress • Succinate; saturated FA • FADH2 + Fe3+ FADH • + H+ + Fe2+ • Fe2+ + H2O2Fe3+ + OH- + OH• • FADH2 more completely reduces UQ than does NADH Acyl-CoA Acyl-CoA FADH2 FAD UQ O2 Acyl-CoA dehydrogenase Acyl-CoA oxidase ETF:QO oxidoreductase FADH2 FAD UQH2 H2O2 Didehydroacyl-CoA Didehydroacyl-CoA

  11. Free fatty acids from triglycerides • FFA cleavage from circulating lipoproteins • Protein/cholesterol carriers: Lipoprotein • Density inversely correlates with lipid • Correlates with cholesterol/FA (except HDL) • VLDL & LDL to IDL • Lipoprotein lipase (LPL) • HDL scavenges cholesterol & facilitates IDL breakdown • Triglycerides are retained in intracellular droplets • Don’t fit in membrane (no phosphate) • Not water soluble

  12. Fatty acid metabolism depends on transport • FAAcyl-CoA Acyl-Carnitine Acyl-CoA Cytoplasm Intermembrane Matrix Working substrate Boron & Boulpaep

  13. Mitochondrial Transport • Carrier protein (FABP) • Long chain acyl-CoA synthetase (LCAS) • Cross outer membrane via porin • Convert to acylcarnitine in intermembrane • Cross inner membrane via carnitine:acylcarnitine transferase • Convert back to acyl-CoA in matrix

  14. Mitochondrial Structure • Principal metabolic engine • Symbiotic bacteria • 6k-370kBP genome • Human: 13 proteins • Dual membrane • ie: two bilayers • Outer membrane highly permeable • Inner membrane highly impermeable

  15. Mitochondrial Matrix • Highly oxidative environment • Unique proton gradient • High pH (8), negative (-180 mV), ~18 kJ/mole • H+ actively transported out of matrix • H+ leak back as H+PO4 2- • Capture gradient energy for ATP synthesis • H+ ATPase pump • ADP-ATP antiporter • Other proton co-transporters • Pyruvate, citrate • Glutamate, citruline

  16. CH3 C=O COO- Metabolic Substrates • Sugars • Metabolized in cytoplasm to pyruvate • Co-transported to matrix with H+ • Bound to Coenzyme A as Acetyl-CoA • Fatty acids • To intermembrane space as Acyl-CoA • To matrix as Acyl-carnitine • Metabolized to Acetyl-CoA in matrix • Proteins

  17. Acetyl Coenzyme A • Common substrate for oxidative metabolism • S-linked acetate carrier

  18. The Citric Acid Cycle Acetyl-Coenzyme A CoA These carbons will be removed NADH New carbons Oxaloacetate Citrate Carbon Malate Oxygen Isocitrate Coenzyme A NADH + = Fumarate a -Ketoglutarate FADH 2 Succinate Succinyl CoA CoA NADH + GTP CoA

  19. NAD+ + H++2e- NADH DE0=-0.32V ½O2+2 H++ 2e- H2O DE0=0.82V NADH + H+ + ½ O2 NAD+ +H2O Electron transport • Couple NADH/FADH2 electrons to H+ export • Ideally this completes • Electron leakage

  20. KEGG pathway • Enzyme Commission (EC) number • Hierarchical • Function-centric nomenclature • Compare • Gene Ontology (GO) ID • Entrez RefSeq • UniProt ID Metabolite KEGG http://www.genome.jp/kegg/pathway.html

  21. NAD+ FAD NADH FADH2 Cyclic redox reactions Oxidized CoQ/ubiquinone Cyto-C3+ O2 dihydroubiquinone Cyto-C2+ H2O Reduced NAD+  NADH E0 = -0.32V FAD FADH2 E0 = -0.22V Ubuquinone E0 = 0.10V Cytochrome C E0 = 0.22V O2  H2O E0 = 0.82V You can only have this progressive redox process if molecular position is carefully controlled

  22. Proton ATPase/Complex V • ATP driven proton pump • “Reversible” • Couples H+ gradient to ATP synthesis

  23. Fatty acid/carbohydrate oxidation • Oxygen • CnH2n + 3/2 n O2n CO2+ n H2O • CnH2nOn +n O2 n CO2 + n H2O • Respiratory Quotient CO2/O2 • 0.67 Fatty acids • 1.00 Carbohydrates • Adenine electron transporters • 6-C glucose6 NADH + 2 FADH2 (3:1) • 16-C FA  32 NADH + 16 FADH2 (2:1) • Redox chemistry differs for FA/CHO

  24. Muscle substrate utilization • Rest: fatty acids • Active: glycolysis • Recovery: • Pyruvate oxidation • Gluconeogenesis

  25. Role of mass action in flux control • Diffusion • J = D ∂f/∂x (greater flux down a steeper gradient) • ∂f/ ∂t= ∂J/∂x • Kinetics • d[P]/dt = k[S] (1st order) • d[P]/dt = Vmax [S]/(Km + [S]) (Michaelis-Menten) • d[P]/dt = k [S1][S2] (2nd order)

  26. Mass action in glycolysis • Diffusion • Substrate consumption increases gradient • Increased gradient accelerates mass flow • Kinetics • G+ATPG6p d[G6p]dt = k1[G][ATP]≈k[G] • G6pF6p d[F6p]/dt = k2[G6p] • F6p+ATPF1,6p <etc> • F1,6pG3p+DAp • DApG3p

  27. Mitochondrial substrate dependence • More ADPfaster ATP • Discharge proton gradient • Lower ETC resitsance • More NADfaster • Faster NADH • Greater ETC input Wu &al 2007

  28. Role of allosteric regulation • Allosteric • Binding to other-than-active site changes enzyme kinetics • Vmax or kM • Many metabolic enzymes are regulated by downstream products • Phosphofructokinase • Citrate inhibits • ADP activates • Gylcogen synthase Allosteric ADP binding site Active site PDB:3PFK

  29. G6P regulation of GS • Allosteric conformational change Without G6P Less active With G6P More active Baskaran et al. 2010

  30. Role of post-translational regulation • Chemical modification of enzymes alters activity • Phosphorylation • Ribosylation, acylation, SUMOylation, etc • Integrative response to complex conditions • Insulin • Insulin  IRPI3KGLUT4 translocation glucose uptake • PI3KPKB--|GSK--|GS

  31. PKA +GP via phosphorylase kinase -GS -PP1 via G-subunit PKB +GS via GSK +PP1 via G-subunit Phospho-regulation of glycogen • PP1 • +GS • -GP Activates Inhibits PK GP GP Glycogen Synthesis PKA PKB PP1-G PP1 PP1 PP1-G GS GS GSK3

  32. AMP kinase • Allosterically activated by AMP • Adenylate kinase: 2 ADP  AMP + ATP • ADP levels insensitive to energy state PFKglycolysis --|GSGlyconeogenesis --|ACCMalonyl CoA--|CPTFA oxidation --|ACClipogenesis TSC2--|mTOR…protein synthesis --|HMGCoAcholesterol synthesis

  33. Summary • Sources of ATP • Creatine • Gylcolysis: GG3p2OPA • Lipolysis: acyl-CoAoxoacyl-CoA • Citric Acid Cycle/Electron Transport Chain • AcCoACitrate...Oxaloacetate • Rate control by • Mass action • Allosteric feedback • Hormonal control

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