1. Glycolysis Anaerobic degradation of glucose to yield lactate or ethanol and CO 2

2. Learning Objectives Sequence of Reactions –Metabolites –Enzymes Enzyme Mechanisms Energetics Regulation

3. Overview of Glycolysis Glucose (C 6 ) —> 2 Pyruvate (C 3 ) 2 ADP + 2 P i —> 2 ATP

4. Figure 15-1 Glycolysis

5. Stage I of Glycolysis (Energy Investment) 2X

6. Summary of Stage I Glucose + 2 ATP ——> 2 GA3P + 2 ADP + 2 H +

7. Stage II of Glycolysis (Energy Recovery) Substrate Level Phosphorylation —> Serine, Cysteine and Glycine —> Aromatic Amino Acids —> Alanine

8. Summary of Stage II 2 GA3P + 2 NAD + + 4 ADP + 2 P i 2 Pyruvate + 2 NADH + 2 H + + 4 ATP

9. Summary of Glycolysis Glucose + 2 NAD + + 2 ADP + 2 P i 2 Pyruvate + 2 NADH + 2 H + + 2 ATP NOTE: NAD + must be regenerated!

10. Reactions of Glycolysis Stage I

11. Hexokinase (First Use of ATP) NOTE: Lack of Specificity  G o’ (kJ/mol)  G (kJ/mol) Glucose + P i  G-6-P + H 2 O 13.8 20.5 ATP + H 2 O  ADP + P i -30.5 -54.4 Glucose + ATP  G-6-P + ADP -16.7 -33.9

12. Page 489 Role of Mg 2+

13. Figure 15-2 Substrate-induced Conformational Changes in Yeast Hexokinase

14. Results of Conformational Change Formation of ATP binding site Exclusion of water Increased nucleophilicity of CH 2 OH Proximity effect

15. Regulation of Hexokinase Inhibition by glucose-6-P Impermeability

16. Hexokinase versus Glucokinase Hexokinase (all tissues) –Non-specific –K M = ~100 µM –Inhibited by glucose-6-P Glucokinase (primarily in liver) –Specific –K M = ~10 mM –Not inhibited by glucose-6-P

17. Functional Rationale Most tissues: metabolize blood glucose which enters cells –Glc-6-P impermeable to cell membrane –Product inhibition Liver: maintain blood glucose –High blood glucose: glycogen –Low blood glucose: glycolysis

18. Figure 22-4 Hexokinase versus Glucokinase

19. Metabolism of Glucose-6-P Regulation!

20. Phosphoglucose Isomerase  G o’ (kJ/mol)  G (kJ/mol) Glucose-6-phosphate  Fructose-6-phosphate 2.2 -1.4

21. Reaction Mechanism of Phosphoglucose Isomerase

22. Figure 15-3 part 1 Reaction Mechanism of Phosphoglucose Isomerase (Substrate Binding)

23. Figure 15-3 part 2 Reaction Mechanism of Phosphoglucose Isomerase (Acid-Catalyzed Ring Opening)

24. Figure 15-3 part 3 Reaction Mechanism of Phosphoglucose Isomerase (Formation of cis-enediolate Intermediate)

25. Figure 15-3 part 4 Reaction Mechanism of Phosphoglucose Isomerase (Proton Transfer)

26. Figure 15-3 part 5 Reaction Mechanism of Phosphoglucose Isomerase (Base-Catalyzed Ring Closure)

27. Figure 15-3 part 1 Reaction Mechanism of Phosphoglucose Isomerase (Product Release)

28. Phosphofructokinase (Second Use of ATP) NOTE: bisphosphate versus diphosphate  G o’ (kJ/mol)  G (kJ/mol) F-6-P + P i  F-1,6-bisP + H 2 O 16.3 36.0 ATP + H 2 O  ADP + P i -30.5 -54.4 F-6-P + ATP  F-1,6-bisP + ADP -14.2 -18.8

29. Characteristics of Reaction Catalyzed by PFK Rate-determining reaction Reversed by Fructose-1,6-bisphosphatase Mechanism similar to Hexokinase

30. Regulatory Properties of PFK Main control point in glycolysis Allosteric enzyme –Positive effectors AMP Fructose-2,6-bisphosphate –Negative effectors ATP Citrate

31. Page 558  - D -Fructose-2,6-Bisphosphate

32. Formation and Degradation of  - D -Fructose-2,6-bisP High glucose Low glucose

33. Aldolase 456456 123123 Carbon # from glucose  G o’ (kJ/mol)  G (kJ/mol) F-1,6-bisP  GAP + DHAP 23.8 ~0

34. Figure 15-4 Mechanism of Base-Catalyzed Aldol Cleavage NOTE: requirement for C=O at C2 Rationale for Phosphoglucose Isomerase

35. Enzymatic Mechanism of Aldolase

36. Figure 15-5 part 1 Enzymatic Mechanism of Aldolase (Substrate Binding)

37. Figure 15-5 part 2 Enzymatic Mechanism of Aldolase (Schiff Base (imine) Formation)

38. Figure 15-5 part 3 Enzymatic Mechanism of Aldolase (Aldol Cleavage)

39. Figure 15-5 part 4 Enzymatic Mechanism of Aldolase (Tautomerization and Protonation)

40. Figure 15-5 part 5 Enzymatic Mechanism of Aldolase (Schiff Base Hydrolysis and Product Release)

41. Triose Phosphate Isomerase  G o’ (kJ/mol)  G (kJ/mol) DHAP  GAP 7.5 ~0

42. Part 494 Enzymatic Mechanism of Triose Phosphate Isomerase

43. Part 494 Transition State Analog Inhibitors of Triose Phosphate Isomerase

44. Figure 15-7 Schematic Diagram of the First Stage of Glycolysis

45. Summary of Stage I Glucose + 2 ATP ——> 2 GA3P + 2 ADP + 2 H +

46. Reactions of Glycolysis Stage II

47. Glyceraldehyde-3-P Dehydrogenase GAPDH 3,4 2,5 1,6  G o’ (kJ/mol)  G (kJ/mol) GAP + NAD+ H 2 O  3-PG + NADH + H+ -43.1 36.0 3PG + P i  1,3-BPG + H 2 O 49.4 -54.4 GAP + NAD+ + P i  1,3-BPG + NADH + H+ 6.3 -18.8

48. Acylphosphate

49. Enzymatic Mechanism of Glyceraldehyde-3-P Dehydrogenase

50. Figure 15-9 part 1 Enzymatic Mechanism of Glyceraldehyde-3-P Dehydrogenase (Substrate Binding)

51. Figure 15-9 part 2 Enzymatic Mechanism of Glyceraldehyde-3-P Dehydrogenase (Thiol Addition)

52. Figure 15-9 part 3 Enzymatic Mechanism of Glyceraldehyde-3-P Dehydrogenase (Dehydrogenation)

53. Figure 15-9 part 4 Enzymatic Mechanism of Glyceraldehyde-3-P Dehydrogenase (Phosphate Binding)

54. Figure 15-9 part 5 Enzymatic Mechanism of Glyceraldehyde-3-P Dehydrogenase (Product Release)

55. 2,3-bisphosphoglycerate Rxn #8 Rxn #7 Rxn #6 Rxns #1-5 Hemoglobin regulation Pyruvate kinase Pyruvate Rxn #9 Rxn #10

56. Glycolysis deficiencies affect oxygen delivery

57. Phosphoglycerate Kinase Formation of first ATPs Substrate-level Phosphorylation

58. Figure 15-10 Yeast Phosphoglycerate Kinase

59. Coupled Reactions       G = ~0

60. Substrate Channeling

61. Phosphoglycerate Mutase  G o’ (kJ/mol)  G (kJ/mol) 3-PGA  2-PGA 4.4 ~0

62. Page 500 Phosphohistidine Residue in Phosphoglycerate Mutase

63. Enzymatic Mechanism of Phosphoglycerate Mutase

64. Figure 15-12 part 1 Enzymatic Mechanism of Phosphoglycerate Mutase (Substrate Binding)

65. Figure 15-12 part 2 Enzymatic Mechanism of Phosphoglycerate Mutase (Phosphorylation of Substrate)

66. Figure 15-12 part 3 Enzymatic Mechanism of Phosphoglycerate Mutase (Phosphorylation of Enzyme)

67. Figure 15-12 part 4 Enzymatic Mechanism of Phosphoglycerate Mutase (Product Release)

68. Enolase Formation of “high energy” intermediate Inhibition by F –  G o’ (kJ/mol)  G (kJ/mol) 2-PGA  PEP -3.2 -2.4

69. Pyruvate Kinase Formation of second ATPs Substrate-level Phosphorylation  G o’ (kJ/mol)  G (kJ/mol) PEP + H 2 O  Pyruvate + P i -61.9 ADP + P i  ATP + H 2 O 30.5 PEP + ADP  Pyruvate + ATP -31.4 -16.7

70. Figure 15-13 Enzymatic Mechanism of Pyruvate Kinase

71. Figure 15-14 Hydrolysis of PEP

72. Regulatory Properties of Pyruvate Kinase Secondary control point in glycolysis Allosteric enzyme –Positive effectors ADP Fructose-1,6-bisphosphate –Negative effectors ATP (energy charge) Acetyl-Coenzyme A

73. Figure 15-15 Summary of Second Stage of Glycolysis

74. Summary of Stage II 2 GA3P + 2 NAD + + 4 ADP + 2 P i 2 Pyruvate + 2 NADH + 2 H + + 4 ATP

75. Summary of Glycolysis Glucose + 2 NAD + + 2 ADP + 2 P i 2 Pyruvate + 2 NADH + 2 H + + 2 ATP NOTE: NAD + must be regenerated!

76. Figure 15-16 Metabolic Fates of Pyruvate

77. Recycling of NADH Anaerobic Fate of Pyruvate

78. Role of Anaerobic Glycolysis in Skeletal Muscle

79. Homolactate Fermentation

80. Page 505 Lactate Dehydrogenase

81. Mechanism of Lactate Dehydrogenase

82. Summary of Anaerobic Glycolysis Glucose + 2 ADP + 2 P i 2 Lactate + 2 ATP + 2 H 2 O + 2 H +

83. Energetics of Fermentation Glucose ——> 2 Lactate Glucose + 6 O 2 ——> 6 CO 2 + 6 H 2 O ∆G o’ = -200 kJ/mol ∆G o’ = -2866 kJ/mol Most of the energy of glucose is still available following glycolysis!

84. Alcoholic Fermentation

85. Figure 15-18 Alcoholic Fermentation

86. Figure 15-18 part 1 Pyruvate Decarboxylase

87. Page 507 Thiamin Pyrophosphate Thiamine = Vitamin B 1

88. Figure 15-20 Mechanism of Pyruvate Decarboxylase

89. Figure 15-20 part 1 Mechanism of Pyruvate Decarboxylase (Nucleophilic Attack)

90. Figure 15-20 part 2 Mechanism of Pyruvate Decarboxylase (CO 2 Elimination)

91. Figure 15-20 part 3 Mechanism of Pyruvate Decarboxylase (Protonation of Carbanion)

92. Figure 15-20 part 4 Mechanism of Pyruvate Decarboxylase (Product Release)

93. Figure 15-18 part 2 Alcohol Dehydrogenase

94. Page 509 Mechanism of Alcohol Dehydrogenase

95. Regulation of Glycolysis and Gluconeogenesis

96. Table 15-1 Free Energy Changes of Glycolytic Reactions

97. Figure 15-21 Diagram of Free Energy Changes in Glycolysis

98. Regulatory Properties of Hexokinase Inhibition by glucose-6-P

99. Metabolism of Glucose-6-P Regulation!

100. Regulatory Properties of Phosphofructokinase Main control point in glycolysis

101. Figure 15-23 Regulation of Phosphofructokinase

102. Regulatory Properties of Pyruvate Kinase Secondary control point in glycolysis Allosteric enzyme –Positive effectors ADP Fructose-1,6-bisphosphate –Negative effectors ATP (energy charge) Acetyl-Coenzyme A

103. Gluconeogenesis

104. Necessity of Glucose-6-P and Glucose

105. Glycolysis and Gluconeogenesis

107. Figure 16-21 Glycolysis and Gluconeogenesis

108. Figure 16-21 Glycolysis and Gluconeogenesis

109. Coordinated Control of Glycolysis and Gluconeogenesis