Lecture 15 –Chapt 16 Glycolysis

1. Lecture 15 –Chapt 16 Glycolysis “Living organisms, like machines, conform to the law of conservation of energy, and must pay for all their activities in the currency of catabolism.” Ernest Baldwin Dynamic Aspects of Biochemistry Louie Pasteur’s scientific investigations into fermentation of grape sugar were pioneering studies of glycolysis.

2. Essential Question • What is the chemical basis and logic for this central pathway of metabolism; that is, how does glycolysis work?

3. OUTLINE • Glycolysis • - First phase - Reactions 1-5 (no ATP generated) • - Second Phase – Reactions 6-10 (generation of ATP) • Fate of pyruvate under anaerobic conditions - In yeast: Alcohol fermentation • In other microorganisms and exercising muscle: Lactic acid fermentation • Feeder pathway to glycolysis (other sugars can also enter the glycolytic pathway) • Regulation of glycolysis • Glucose transporters

4. Glucose is the body primary fuel supply

5. Metabolic pathways are compartmentalized within cells Glycolysis pathway common to both prokaryotes and eukaryotes. In eukaryotes, glycolysis takes place in cytoplasm Phase 1 Phase 2

6. 1st Stage of Glycolysis Uses 2 molecules of ATP/glucose but makes no ATP. Traps and prepares glucose for following oxidation steps Glucose is converted to GAP 5 steps: 1. Phosphorylation 2. Isomerization 3. Second phosphorylation 4. Cleavage into two 3-carbon molecules 5. Isomerization of DHAP to GAP

8. Glycolysis: Reaction 1 - Phosphorylation Hexokinase Traps Glucose in the Cell and Begins Glycolysis

9. Glucose is kept in the cell by phosphorylation to glucose-6-phosphate

10. Hexokinase

11. Glycolysis: Reaction 2 – Isomerization by phosphoglucose isomerase

12. Glycolysis: Reaction 3- Second Phosphorylation Phosphofructokinase (PFK) Committed step

13. Glycolysis: Reactions 4 – Cleavage of one 6-carbon to two 3-carbon molecules

14. Glycolysis: Reactions 5 - end of 1st phase of glycolysis

15. Second Stage of Glycolysis The three carbon units are oxidized to pyruvate, generating 4 molecules of ATP/glucose and 2 NADH/glucose GAP is converted to pyruvate: 5 steps: 6. Oxidation of GAP to 1,3BPG 7. Phosphorylation of ADP 8. Mutase 9. Dehydration by Enolase 10. Phosphorylation giving pyruvate

16. Glyceraldehyde 3-phosphate

17. Glycolysis Reaction 6 - GAP is oxidized to 1,3-BPG High-energy phosphate Precursor to 2,3BPG in RBC

18. ∆G°’ = -50 kJ mol-1 ∆G°’ = ~ +56 kJ mol-1

19. Coupling of the two processes allows the conservation of energy released by oxidation Uncoupled processes Coupled processes

20. High-energy phosphate compounds ATP ADP

21. Glycolysis: Reaction 7 (ATP producing reaction) Substrate-level phosphorylation 2 ATP consumed in the 1st phase of glycolysis (Reactions 1 and 3) are ‘paid off’ in the this step of the 2nd phase of glycolysis.

22. Coupling of Reactions 6 and 7 Preparative step for ATP production in the following step GAP 1,3-BPG 3 PG Step 6 High energy intermediate phosphate compound ∆G°’ = +6.3 kJ/mol Actual ATP production Step 7 ∆G°’ = -18.9 kJ/mol ATP The formation of the high energy intermediate compound 1,3-BPG (in step 6) is essential as a preparation for the ATP production (in step 7).

23. Glycolysis: Reaction 8 – Phosphoglycerate Mutase (Note: The term Mutase is applied to those enzymes that catalyze migration of functional groups from one position to another on the same substrate molecule)

24. Glycolysis: Reaction 9 - Dehydration of 2-PG ΔG°′ = +1.8 kJ/mol

25. High-energy phosphate compounds ATP ADP

26. Glycolysis: Reaction 10 - ATP synthesizing reaction (Regulated step) Pyruvate kinase ΔG°′ = -31.7kJ/mol Note: Steps 9 and 10 are coupled (just like steps 6 and 7)

27. The net reaction for glycolysis is ∆G°’= 96 kJ mol-1

28. The free energies of the reactions of glycolysis under standard-state conditions.

29. The free energies of the reactions of glycolysis under actual intracellular conditions in erythrocytes. ∆G = -96 kJ mol-1

30. Fate of pyruvate Add mitochondria Add workingmuscle Addbottle of Moet et Chandon

31. Alcoholic Fermentations are a means of Oxidizing NADH Uses Thiamine pyrophosphate (TTP) Pork and legumes are a good source of thiamin (vit B1)

32. Lactic Acid Fermentations are a means of Oxidizing NADH .

33. Entry points in glycolysis for galactose and fructose.

34. Fructose enters the glycolytic pathway in the liver through the fructose 1-phosphate pathway.

35. Fructose can be converted to F-6P by hexokinase. hexokinase

36. Galactose enters the glycolytic pathway in the liver through the glucose 6-phosphate pathway.

37. Galactose in Glycolysis?

38. Lactose – From Mother’s Milk to Yogurt – and Lactose Intolerance

40. Regulation of Glycolysis • hexokinase • phosphofructokinase • pyruvate kinase Enzymes catalyzing irreversible reaction in metabolic pathways are potential control sites.

41. Hexokinase versus Glucokinase Muscle contains Hexokinase I while liver contains Hexokinase IV (Glucokinase). This is to adapt to the different roles of muscle and liver. Vmax 2 Km (hexokinase) = 0.03 mM Km (glucokinase) = 10 mM

42. Regulation of Glycolysis • hexokinase • phosphofructokinase • pyruvate kinase The pacemaker of glycolysis Enzymes catalyzing irreversible reaction in metabolic pathways are potential control sites.

43. Regulation of Allosteric Enzymes by feedback Mechanism Negative Feedback Inhibition X e1 e2 e3 e4 e5 A  B  C  D  E  F Effector Committed step

44. Glucose-6-P is common to several metabolic pathways

45. Regulation of Phosphofructokinase In muscles: PFK In liver: + + +

46. Allosteric Regulation of Phosphofructokinase High [AMP] = = Low [AMP] ATP is an allosteric inhibitor of PFK AMP is an allosteric activator of PFK

47. Changes in AMP level is a more sensitive indicator of a cell’s energetic state (than changes in ATP or ADP levels) Under most cellular conditions, ATP concentration does not vary over a large range Adenylate kinase ADP + ADP ATP + AMP (one ADP acts as the P donor, the other ADP as the P acceptor)

48. Regulation of Phosphofructokinase In muscles: PFK In liver: + + +

49. Fructose 2,6-biphosphate

50. The activation of phosphofructokinase by fructose 2,6-bisphosphate.