1. Glycolysis Chapter 16 – Voet and Voet 2 nd Edition Wed. September 25, 2002 1. The Glycolytic Pathway 2. The Reactions of Glycolysis 3. Fermentation: The Anaerobic Fate of Pyruvate 4. The Glycolytic Flux 5. Metabolism of Hexoses Other Than Glucose

2. Electron micrograph of yeast cells. Fermentation of glucose to ethanol and CO 2 by yeast has been a useful process since before the dawn of recorded history. Winemaking and bread baking both use this process.

3. The Glycolytic Pathway A. Historical Perspective B. Pathway Overview

4. A. Historical Perspective In years 1854 to 1864, Louis Pasteur established that fermentation is caused by microorganisms. 1897 – Eduard Buchner demonstrated that cell-free yeast extracts can carry out this process. In years 1905 to 1910, Arthur Harden and William Young discovered: –Inorganic phosphate is required for fermentation and is incorporated into fructose-1-6-bisphosphate –A cell-free yeast extract has a nondialyzable heat-labile fraction (zymase) and a dialyzable heat-stable fraction (cozymase). Elucidation of complete glycolytic pathway by 1940 (Gustav Embden, Otto Meyerhof, and Jacob Parnas).

5. Inhibitors were used to study metabolic pathways. For example, addition of fluoride ion to fermenting yeast extracts causes a buildup of 2-phosphoglycerate and 3-phosphoglycerate, glycolytic pathway intermediates. This is caused by inhibition of enolase.

7. Glycolysis Glucose is converted to pyruvate while generating two ATPs. 2 molecules of NAD + are converted to 2 molecules of NADH. The oxidizing power of NAD + must be recycled.

8. B. Pathway overview

9. Pathway Overview There are 10 enzyme-catalyzed reactions considered to occur in two stages –Stage I (reactions 1-5): Preparatory stage where glucose is phosphorylated and cleaved to yield 2 molecules of glyceraldehyde-3-phosphate (GAP). Stage I uses 2 ATPs. –Stage II (reactions 6-10) Payoff stage where 2 GAPs converted to pyruvate and generation of 4 ATPs.

10. Glycolytic Pathway Stage 1 Stage 2

11. 2. The Reactions of Glycolysis Stage I (Preparatory Stage) 1. Hexokinase (first ATP utilization) 2.Phosphoglucose Isomerase (PGI) 3.Phosphofructokinase -1 (PFK-1) (second ATP utilization) 4.Aldolase 5.Triose Phosphate Isomerase (TIM)

13. THE PREPARATORY PHASE Step 1 – Hexokinase

14. (A) Hexokinase: First ATP Utilization Reaction 1 of glycolysis is the transfer of a phosphoryl group from ATP to glucose to form glucose 6-phosphate (G6P).

15. Hexokinase and its complex with glucose (purple molecule). In the enzyme-substrate complex the two lobes (grey and green) swing together to engulf the substrate. This excludes H 2 O from the active site which prevents ATP hydrolysis. Glucose

16. Phosphoglucose Isomerase catalyzes the conversion of G6P to F6P, the isomerization of an aldose to a ketose. Step 2 – Phosphoglucose Isomerase (PGI) [Phosphohexose isomerase]

17. The isomerization of an aldose to a ketose

18. (C) Step 3 - Phosphofructokinase-1: PFK-1 phosphorylates fructose-6- phosphate (F6P) in reaction 3 of glycolysis (second ATP utilization). PFK plays a central role in control of glycolysis because it catalyzes one of the pathway’s rate-determining reactions.

19. (C) Step 3 - Phosphofructokinase 1: Second ATP utilization

20. D. Step 4 - Aldolase Aldolase catalyzes cleavage of fructose- 1,6-bisphosphate (FBP) in reaction 4 of glycolysis. This forms two trioses –Glyceraldehyde-3-phosphate (GAP) –Dihydroxyacetone phosphate (DHAP).

21. Step 4 - Aldolase. Aldol cleavage of FBP to form two Trioses (GAP and DHAP) Note that the atom numbering system changes. Atoms 1, 2, and 3 of glucose become atoms 3,2, and 1 of DHAP. Atoms 4, 5, and 6 become atoms 1, 2, and 3 of GAP.

22. Mechanism of base-catalyzed aldol cleavage. Enzymatic reaction

23. Aldolase Enzymatic mechanism of Class I aldolase.

24. (E) Step 5 - Triose Phosphate Isomerase (TIM) Only GAP continues along the glycolytic pathway.

25. Fate of the carbon atoms of glucose in the formation of glyceraldehyde-3-phosphate.

26. Ribbon diagram of TIM in compex with its transition state analog 2-phosphoglycolate. The flexible loop (residues 168 -177) (light blue) makes a hydrogen bond with the phosphate group of the substrate. Removal of this loop by mutagenesis does not impair substrate binding but reduces catalytic rate by 10 5 fold.

27. Stage II - payoff phase 6. Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) first "High-energy" intermediate formation. 7. Phosphoglycerate Kinase (PGK): First ATP Generation. 8. Phosphoglycerate Mutase (PGM). 9. Enolase: second "High-energy" intermediate formation. 10. Pyruvate Kinase (PK): Second ATP generation.

28. (F) Step 6 - Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH): First “High-Energy” Intermediate Formation.

29. Glyceraldehyde-3-phosphate dehydrogenase reaction

30. Enzymatic Mechanism of GAPDH

31. (G). Step 7 - Phosphoglycerate Kinase (PGK): First ATP Generation.

32. Phosphoglycerate Kinase - Upon substrate binding, the two domains of PGK swing together to permit substrates to react in a water-free environment as occurs with hexokinase. 3PG

33. Mechanism of the PGK reaction.

34. The energetics of the overall GAPDH-PGK reaction pair.

35. (H). Step 8 - Phosphoglycerate Mutase (PGM).

36. Reaction Mechanism of PGM (1) Catalytic amounts of 2,3-Bisphosphoglycerate Are required for enzymatic activity.

37. (2) Incubation of the enzyme with catalytic amounts of 32 P-labeled 2,3-BPG yields a 32 P-labeled enzyme.

38. PGM (step 8) reaction mechanism

39. Glycolysis influences oxygen transport 2,3-BPG binds to deoxyhemoglobin and alters the oxygen affinity of hemoglobin. Erythrocytes synthesize and degrade 2,3-BPG by a detour from the glycolytic pathway.

40. Lower [BPG] in erythrocytes resulting from hexokinase- deficiency results in increased hemoglobin oxygen affinity. [BPG]

41. (I) Step 9 - Enolase: Second “High- Energy” Intermediate Formation. (Dehydration reaction)

42. Reaction mechanism of enolase.

43. (J) Step 10 - Pyruvate Kinase (PK) : Second ATP Generation.

44. Pyruvate Kinase Tautomerization of enolpyruvate to pyruvate.