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 C 6 H 12 O 6 – End 2xC 3 H 3 O 3 – Added 0xO – Lost 6xH – Gained 2xNADH, 2xATP NADH ATP pyruvate kinase GAPDH phosphoglycerate 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 pyruvate 2-Hydroxyethyl- Thiamine diphosphate S-acetyldihydro- lipoyllysine Acetyl-CoA

7. Carbohydrate metabolism depends on transport H+, pyruvate cotransporter Halestrap & Price 1999 Major Facilitator Superfamily Monocarboxylate transporter 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”

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

10. Reactive oxygen Acyl-CoA Didehydroacyl-CoA FAD FADH2 Acyl-CoA dehydrogenase Acyl-CoA Didehydroacyl-CoA O2 H2O2 Acyl-CoA oxidase UQ UQH 2 FADH 2 oxidative stress – Succinate; saturated FA – FADH 2 + Fe 3+  FADH + H + + Fe 2+ – Fe 2+ + H 2 O 2  Fe 3+ + OH - + OH FADH 2 more completely reduces UQ than does NADH FADH 2 FAD ETF:QO oxidoreductase

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 CytoplasmIntermembraneMatrixWorking 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 + PO 4 2- Capture gradient energy for ATP synthesis – H + ATPase pump – ADP-ATP antiporter Other proton co-transporters – Pyruvate, citrate – Glutamate, citruline

16. 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 CH 3 C=O COO -

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

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

19. Electron transport Couple NADH/FADH2 electrons to H+ export – Ideally this completes – Electron leakage NADH + H + + ½ O 2 NAD + +H 2 O NAD + + H + +2e - NADH  E 0 =-0.32V ½O 2 +2 H + + 2e - H 2 O  E 0 =0.82V

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

21. Cyclic redox reactions Oxidized Reduced NADH FADH 2 NAD + FAD CoQ/ubiquinone dihydroubiquinone Cyto-C 3+ Cyto-C 2+ O2O2 H2OH2O NAD+  NADH E 0 = -0.32V FAD  FADH 2 E 0 = -0.22V Ubuquinone E 0 = 0.10V Cytochrome C E 0 = 0.22V O 2  H 2 O E 0 = 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 – C n H 2n + 3/2 n O 2  n CO 2 + n H 2 O – C n H 2n O n +n O 2  n CO 2 + n H 2 O – Respiratory Quotient CO2/O2 0.67 Fatty acids 1.00 Carbohydrates Adenine electron transporters – 6-C glucose  6 NADH + 2 FADH 2 (3:1) – 16-C FA  32 NADH + 16 FADH 2 (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 ∂  /∂x (greater flux down a steeper gradient) – ∂  / ∂t= ∂J/∂x Kinetics – d[P]/dt = k[S] (1 st order) – d[P]/dt = Vmax [S]/(Km + [S]) (Michaelis-Menten) – d[P]/dt = k [S 1 ][S 2 ] (2 nd order)

26. Mass action in glycolysis Diffusion – Substrate consumption increases gradient – Increased gradient accelerates mass flow Kinetics – G+ATP  G6p d[G6p]dt = k 1 [G][ATP]≈k[G] – G6p  F6p d[F6p]/dt = k 2 [G6p] – F6p+ATP  F1,6p – F1,6p  G3p+DAp – DAp  G3p

27. More ADP  faster ATP – Discharge proton gradient – Lower ETC resitsance More NAD  faster – Faster NADH – Greater ETC input Mitochondrial substrate dependence 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 PDB:3PFK Allosteric ADP binding site Active site

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

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