Cell Respiration & Fermentation

1. Cell Respiration & Fermentation Chapter 9

2. 4 Major Steps in Metabolism • Step 1 • Glycolysis – glucose to 2 pyruvate • Production of 2 ATP & 2 NADH • Remember if no oxygen is present then steps 2 to 4 do not occur and fermentation takes place • Step 2 • Pyruvate Dehydrogenase Complex • Production of Acetyl CoA, NADH & CO2 • Step 3 • Krebs cycle • Complete oxidation of your food • Production of 1 ATP, 3NADH, 1FADH2 & 2 CO2 • Step 4 • Electron Transport Chain – ETC • Production of proton gradient by electron “hot potato” • Production of ATP by ATP synthase • Consumption of O2 and production of H2O

3. Cellular location Cytoplasm Two Phases Preparatory phase Invest 2 ATP Payoff phase Get out 4 ATP Get out 2 NADH End with two molecules of pyruvate Ancient pathway evolved before photosynthesis Anaerobic pathway Most heterotrophic organism on earth use this pathway Same 10 steps Same enzymes Glycolysis

4. Glycolysis Prep Phase Enzymes: 1) Hexokinase 2) Phosphoglucose isomerase 3) PFK-1 Phosphofructokinase-1 4) Fructose-bis-phosphaste aldoalse 5) Triose phosphate isomerase TIM

5. Glycolysis – Payoff Phase Enzymes: 6) Glyceraldehyde – 3- phosphate dehydrogenase 7) Phosphoglycerate kinase 8) Phosphoglycerate mutase 9) Enolase 10) Pyruvate kinase

6. Glycolysis and ATP Production • All ATP made during glycolysis requires no oxygen • All ATP made is during glycolysis is known as substrate level phosphorylation

7. Regulation of Glycolysis • G6P has other choices • Glycogen synthesis • Pentose phosphate pathway • The production of F1,6BP commits glucose to glycolysis • PFK-1 is the major regulation point of glycolysis

8. PFK – 1 Regulation • Several regulation sites ADP AMP ATP F1,6BP ADP F6P ATP + + Citrate F2,6BP

9. PFK – 1 Regulation • How does it makes sense that an increase in substrate would decrease the reaction rate • Feedback inhibition ATP is also an allosteric inhibitor

10. F2,6BP • Fructose 2, 6 bisphosphate is not fructose 1, 6 bisphosphae • Fructose 2, 6 bisphosphate is not a substrate of PFK – 1 • Fructose 2, 6 bisphosphate is a regulator of PFK – 1 • Fructose 2, 6 bisphosphate is made by an enzyme • Phosphofructokinase – 2 (PFK -2)

11. Glucagon & Insulin Regulation of Glycolysis • When blood sugar levels drop glucagon is releases from the pancreases • Glucagon activates a standard G – protein pathway which activates PKA • PKA phosphorylates and inactivates PFK -2 • F 2,6 BP levels fall • PFK -1 is inactive • No glycolysis • Insulin has the opposite effect

12. PFK – 2 Regulation

13. Glycolysis and Cancer • Glucose metabolism is 10X faster in some tumors • Tumor cells commonly experience hypoxia • Because initially they lack an extensive capillary network • Hypoxia induced transcription factor HIF-1 • Stimulates expression of eight glycolytic genes

14. Fermentation • Earth earths conditions did not have oxygen • Oxygen levels arouse as photosynthesis evolved, which was after glycolysis • If NADH levels increase in the cell this will inhibit glycolysis • Therefore, fermentation pathways evolved to compensate for the increase in NADH levels • Solution was to oxidize pyruvate, decrease levels of NADH, increase NAD+ and allow glycolysis to continue

15. Fermentation If no oxygen is present fermentation pathways will proceed You learned in chapter 7 about endosymbiosis and mitochondria. Once that happened and enough oxygen was present aerobic respiration could take place which has three additional steps

16. Fermentation Humans Homolactic acid fermentation

17. Fermentation Yeast

18. Step 2 - PDC • Entry of pyruvate into the mitochondria requires a specific transporter is an active transport process

19. PDC Regulation • Negative & Positive Regulation – • Pyruvate processing stops when PDC is Phosphorylated • Phosphorylation changes the shape of the enzyme complex making the binding of substrate not possible • High concentration of NADH and acetyl CoA also inhibit the enzyme • High concentration of NAD+, CoA or AMP increases reaction rate of PDC • Large amounts of pyruvate inhibit the complex • Low amounts of pyruvate stimulate the complex

20. Step 3 – Krebs

21. Step 3 • Involves 8 small carboxylic acids • R-COOH • The energy released by the oxidation of one molecule of acetyl CoA is conserved by the production of • 3 NADH • 1 FADH2 • 1 ATP

22. Regulation of Krebs

23. Purpose of NADH and FADH2 • The electron transport chain transfers electrons from NADH to other electron carriers • The electrons end up on various molecules • However, electron movement is associated with making a proton gradient • Proton gradient is responsible for ATP production

24. Step 4 – ETC • Basic idea is that electrons pass from a molecule with lower electronegativity to one with higher electronegativity • Electrons held more tightly as they pass through the chain • As electrons pass through the chain they go from higher to lower potential energy • The energy released by electron transfer is conserved in the production of a proton gradient

25. Getting Pyruvate into the Mitochondria

26. Details of the ETC

27. ETC in Humans

28. More affinity for e- Less affinity for e-

29. Anaerobic Metabolism • Oxygen is the most effective electron acceptor due to its high electronegativity • Cells that do not use oxygen as an electron acceptor can still use and ETC to generate ATP • However, the final electron acceptor is • NO31- • SO42-

30. Chemiosmotic Hypothesis • Indirect link between electron transport and ATP production • This idea proposed by Peter Mitchell in 1961 was rejected by researcher who believed that a component of the ETC phosphorylated ADP • Substrate level phosphorylation

31. Chemiosmotic Hypothesis • Researchers could isolate different proteins in the inner membrane of the mitochondria • These proteins would be exposed to ATP or ADP and Pi • Only one protein was able to perform ATP hydrolysis or ADP + Pi production • This isolated protein was inserted into an artificial vesicle with Bacteriorhodopsin

32. ATPProduction • ATP production could occur in the absence of an ETC • ATP production depends of the presence of a proton gradient

33. How much ATP is made? • These are only guidelines but the rule is: • 1 NADH = 3 ATP • 1 FADH2 = 2 ATP • In any given cell type these numbers can vary slightly • How are these molecules linked directly to ATP production

34. Energy Book Keeping Remember you make 2 acetyl coA’s from glucose so Krebs cycle turns twice

35. Is Respiration Coupled to ATP Synthase?

36. Coupling • How could you uncouple the ETC from the production of ATP • What if you treated a cell with a weak hydophobic acid such as FCCP or DNP

37. DNP Caused Uncoupling

38. Methods of ATP Production • Substrate level phosphorylation • Glycolysis & Krebs • Enzyme transfers the phosphate group from a phosphorylated substrate to ADP • Oxidative phosphorylation • The phosphorylation of ADP is linked to the consumption of oxygen and the creation of a proton gradient

39. Details of the ETC • Structural studies to date confirm • Complexes I and IV pass protons directly through a sequence of electron carriers • Exact route remains unknown • Best understood interaction is in complex III • Q cycle • Q takes electrons from complex I or II • Q carries two electrons and two protons • Q drops off electrons and protons to outer side of inner membrane in interactions with complex III

40. Details of the ETC • Most proteins in the ETC contain distinct chemical groups which are easily oxidized or reduced (Complexes I, II, III, & IV) • Flavins • Iron-sulfur groups • Heme groups • Ubiquinone or coenzyme Q is an exception and does not have any of these groups

41. Coenzyme Q • Lipid soluble • Carries 2e- & 2H+ from either complex I or II • Drops off electrons with complex III

42. Q Cycle

43. Interconnection of Metabolic Pathways – Lipids • Lipids are broken down in the mitochondria through a series of reactions known as β oxidation • β oxidation breaks lipids apart two carbon units at a time and produces one molecule of acetyl CoA • β oxidation requires water and also produces • 1 FADH2 • 1 NADH • This is per one cycle of beta oxidation so the amount of total NADH & FAHD2 depends on the length of the fat

44. β oxidation NADH FADH2 NADH FADH2 NADH FADH2 NADH FADH2 NADH FADH2 7 Total of 7 NADH Total of 7 FADH2

45. Interconnection of Metabolic Pathways – Proteins Deamination

46. Deamination • Deamination reactions remove the amino group from amino acids • This group is replaced by oxygen which comes from water • The carbon skeletons can then enter into the Krebs cycle at various points • These reactions are reversible • Amination

47. Amination

48. Cellular Yield of ATP