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3. Fatty acid oxidation Also called beta-oxidation –Because the action occurs on the beta-carbon atom

4. Fatty acid oxidation Requires tissues to have mitochondria Reciprocally regulated with glucose oxidation –Fatty acid oxidation inhibits glucose oxidation Consumes a lot of FAD, NAD, CoA –Availability of cofactors is important

5. Transport of FA Albumin Fatty acid binding protein

6. Transport of FA FA needs to be transported from blood into tissues FA is carried in blood on albumin – Several binding sites for FA There are specific transporters for FA –CD36 moves to the cell surface whenever there is a need to take up FA at a rapid rate FA is carried on FABP (fatty acid binding protein) in cytoplasm

7. Trapping of FA FA is trapped by attaching it to CoA This also ‘activates’ the fatty acid (‘tags’ the FA) Requires quite a lot of energy, –ATP is not converted into ADP, but AMP

11. Transport of FA: Mitochondria

12. FA-CoA is oxidized Example: 16C FA-CoA 7 NADH & 7 FADH 2 are produced…. NAD & FAD needed 8 acetyl CoA produced…. CoA needed

14. Cofactor Availability NAD, FAD and CoA –All needed to keep FA oxidation going How are these carriers regenerated? –CoA By entry of acetyl CoA into Krebs Cycle –NAD/FAD By giving cargo to electron transport chain

15. Rate Limiting Enzymes The slowest enzyme in the metabolic pathway determines the overall speed –Rate-limiting step (RLS) –Flux generating step Key points of regulation

16. Enzyme kinetics  At high [substrate], minor changes in [substrate] will not affect the rate of reaction Doubling or halving the [S] isn’t even going to affect the rate [substrate] Rate V max ½ V max KmKm S1S1 S2S2

17. Redfern Station Analogy Imagine the station at peak hour with just one barrier operating –This gate will soon become ‘saturated’ with people –Increasing the number of people doesn’t increase the rate –It is the ‘rate limiting’ step –The point which determines the overall rate at which people get to Uni

18. Changing the Flux There are 3 major ways to regulate this (and metabolic!) pathways –Change the intrinsic activity of the step Make ticket-reading & gate-opening happen faster –Make more gates open Switch them from being ‘off’ to ‘on’ Or change the direction from ‘in’ to out Or bring in a set of gates when you need them –Make and destroy gates according to need Seems crazy!

19. Properties of RLS Irreversible –Need alternative enzymes to ‘go back’ –Not ‘equilibrium’ under physiological conditions –“Committed steps” Saturated with substrate –Low Km or [S] >> Km –Working at Vmax

20. RLS in FA ox? Availability of fatty acids? Cell membrane transport & Trapping? Mitochondrial transport? Carnitine Oxidation? –Activity of enzymes Co-factor availability? Does it depend on the circumstances?

22. Glycolysis Uses carbohydrate (glucose) Wholly cytosolic All cells of the body No requirement for oxygen Very, very fast Very inefficient

23. Glucose

24. Glucose Uptake P glucose Using ATP hexokinase glucose 6-phosphate blood cytoplasm glucose Uptake facilitated by Glucose transporters (GLUTs) GLUT-1 present in all cells all the time GLUT-4 muscle and adipose tissue (the insulin sensitive tissues) GLUT-2 liver and pancreas (blood glucose regulating tissues)

25. Early Glycolysis P Using ATP Phosphofructokinase glucose 6-phosphate P PP fructose 6-phosphate fructose 1,6-bisphosphate PFK Investment of energy giving a bi- phosphorylated symmetrical sugar P P Two molecules of 3-carbon sugar phosphates Splitting to give two 3-carbon molecules

27. Return Phase P Oxidize with NAD Remember two 3-carbon molecules go down the pathway pyruvate Recoup some ATP P P Bring in phosphate P Recoup some ATP Super energy molecule!

29. Overview Total yield is 2 ATP per glucose –And two pyruvate –And two NADH Need to regenerate NAD Fate of the pyruvate –Aerobic –Anaerobic

32. Completing Glycolysis More ATP from oxidation of pyruvate –Need to transport into mitochondria –Oxidize with pyruvate dehydrogenase Need to reoxidise NADH –To maintain the supply of NAD –Shuttle systems available To send NADH electrons/hydrogens into matrix –Lactate production –In yeast, alcohol production –Latter two keep everything cytosolic

34. Regulation Most points reversible Focus on three steps –Hexokinase (G  G6P) Mainly feedback inhibition from G6P –Phosphofructokinase (F6P  F6BP) Strongly affected by ATP/ADP levels But mainly via AMP levels –Pyruvate kinase (last step) ATP/ADP important

35. Energy Charge Large changes in ATP not desirable –Keep ATP at 5 mM Adenylate kinase –Translates small change in ATP to large relative change in AMP –2ADP  ATP + AMP Ratio of adenine nucleotide concentrations often called ‘energy charge’ Strong stimulation of PFK

36. Energy Charge

37. glucose FA FA-CoA OAA pyruvate glycogen G6P pyruvate FA-CoA ac-CoA citrate CO 2 ac-CoA IC 2OG CO 2 GLYCOLYSIS PHOSPHORYLASE BETA-OXIDATION KREBS CYCLE PDH GLUT-4 “CARNITINE” CD36 ICDH OGDH Integration of Catabolism

38. Krebs Cycle TCA cycle, Citric Acid cycle Substrate is acetyl CoA –Fatty acid oxidation and/or glucose oxidation Overall strategy –Completely oxidize acetate carbons to CO 2 –Produce lots of NADH, FADH2, even an ATP –Perform the reaction on a carrier molecule –Regenerate the carrier

41. Regulation Krebs cycle activity is controlled early on –At isocitrate dehydrogenase (ICDH) –alpha-ketoglutarate dehydrogenase (OGDH) ICDH and OGDH are stimulated by rise in Ca 2+ –Such as is found during exercise ICDH & OGDH are also sensitive to NAD levels –Activity is dependent on availability of NAD

43. Important Features During the cycle –2 carbon atoms come in, 2 carbon atoms released Generates –3 NADH, 1 reduced FAD plus a GTP –Each NADH gives 2.5 ATP in oxidative phosphorylation –Each FADH 2 gives 1.5… –So with the GTP, that’s about 10 ATP per acetate Oxaloacetate is not ‘net’ consumed in the cycle – acts as carrier