1. GLYCOLYSIS Student Edition 5/30/13 version
Dr. Brad Chazotte
213 Maddox Hall
Web Site:
Original material only © B. Chazotte
Pharm. 304 Biochemistry
Fall 2014

2. Goals Learn the enzymes and sequence of reactions in glycolysis
Develop an understanding of the chemical “logic” of the glycolysis pathway
Understand the basis and need for redox balance in glycolysis
Learn and understand the control(s) and control points of the glycolysis pathway.
Learn where products of glycolysis can go.
Be aware that other sugars can enter the glycolysis pathway

3. An Energy Conversion Pathway Used by Many Organisms
Glycolysis:
An Energy Conversion Pathway Used by Many Organisms
Almost a universal central pathway for glucose catabolism
The chemistry of these reactions has been completely conserved.
Glycolysis differs among species only in its regulation and in the metabolic fate of the pyruvate generated.
In eukaryotic cells glycolysis takes place in the cell cytosol.

4. The Glycolysis Pathway [Embden-Meyerhof Pathway]
Glycolysis is the sequence of reactions that metabolizes one molecule of glucose to two molecules of pyruvate with the concomitant net production of two molecules of ATP
Glycolysis is an anaerobic process, i.e., it does not require oxygen
Voet, Voet & Pratt 2013 Fig 15.1

5. Overall Reaction of Glycolysis
Glucose + 2NAD+ + 2ADP + 2Pi
2 pyruvate + 2 NADH + 2H+ + 2ATP + 2H2O
Conversion of glucose into pyruvate: G1 = -146 kJ mol-1
Glucose + 2NAD+ 2 pyruvate + 2 NADH + 2H+
Formation of ATP from ADP and Pi G2 = 2 (30.5)= 61 kJ mol-1
2ADP + 2Pi 2ATP + 2H2O
Gs = G1 + G1 = -146 kJ mol kJ mol-1 = -85 kJ mol-1

6. The Glycolysis Pathway
There are three major stages of glycolysis defined (some texts define two):
Trapping and destabilization of glucose (2 ATP used)
Cleavage of 6-carbon fructose to two interconvertible 3-carbon molecules (4 ATP produced)
Generation of ATP

7. Examples of Glucose Metabolic Fates
Major Glucose Utilization Pathways in Cells of Higher Plants and Animals
Catabolism via Pyruvate
Pyruvate
O O -
CH3 C C O
Voet, Voet & Pratt 2013 Fig 15.16
Lehninger 2000 Fig 15.1

8. FERMENTATION
Definition: A general term for the anaerobic degradation of glucose or other organic nutrients to obtain energy conserved in the form of ATP.
Disadvantage: Fermentations produce less energy than complete combustion with oxygen
Advantage: Does not require oxygen. Gives an organism a wider choice of habitats.
TWO EXAMPLES OF FERMENTATION:
Alcohol Fermentation: e.g. the conversion of pyruvate from glycolysis to ethanol in yeast CH3-CH2OH
Lactic Acid Fermentation: e.g. the conversion of pyruvate from glycolysis to lactic acid in skeletal muscle. CH3-CHOH-COO-

9. Reactions of Glycolysis
Berg, Tymoczko & Stryer, 2012 Table. 16.1

10. Schematic of the Glycolysis Pathway
Horton 2-stage
Schematic of the Glycolysis Pathway
1. Trap and destabilize
Hexose stage
2. Cleave 6-C into two 3-C molecules
3. Generate ATP
Triose stage
Berg, Tymoczko & Stryer, 2002 Fig. 16.3

11. Stage 1 of Glycolysis Detail
Berg, Tymoczko & Stryer, 2002 Fig. 16.X

12. Conversion of Glucose by Hexokinase
Hexokinase present in all cells of all organisms
Kinases are enzymes that catalyze the transfer of a phosphoryl group from ATP to an acceptor
carbon numbering
Reaction Purposes:
1. Traps glucose in the cell due to the negative charges on the phosphoryl groups which are ionized at pH 7. Precludes diffusion through the plasma membrane.
2. The attachment of the phosphoryl group renders glucose a less stable molecule and more amenable to further metabolic action.
mechanism
G= kJ/mol
Lehninger 2000 Fig 15.1
Glycolysis Step 1
Horton, 2002 Fig 11.3

13. Hexokinase Structure & Glucose Binding
Yeast Hexokinase
Two lobes move towards each other as much as 8 Å when glucose is bound
Resulting cavity creates a much more nonpolar environment around the glucose molecule which favors the donation of the ATP’s terminal phosphate
Voet, Voet & Pratt , 2008 Fig. 15.2
Berg, Tymoczko & Stryer, 2012 Fig. 16.3

14. Isomerization of Glucose-6-P to Fructose-6-P
G=1.7 kJ/mol
Glycolysis Step 2
Berg, Tymoczko & Stryer, 2012 Chap 16 p. 457

15. Phosphoglucose Isomerase Mechanism
Enzyme active site
Lys?
Glu?
Glycolysis Step 2
Voet, Voet & Pratt Fig. 15.3

16. Phosphorylation of Fructose 6-P
G= kJ/mol
Glycolysis Step 3
Berg, Tymoczko & Stryer, 2012 Chap 16

17. Stage 2 of Glycolysis Berg, Tymoczko & Stryer, 2002 chap 16.

18. Cleavage of Fructose 1,6-biphosphate by Aldolase
G=23.8 kJ/mol
Glycolysis Step 4
Berg, Tymoczko & Stryer, 2012 chap 16 p. 458

19. Aldolase Reaction: Glycolysis Rx #4
Glycolysis Step 4
Voet, Voet & Pratt p. 478

20. Base-catalyzed Aldol Cleavage Mechanism
Glycolysis
Voet, Voet & Pratt 2013 Fig. 15.4

21. Aldolase Mechanism
The cleavage by aldolase of F1,6BP stabilizes the enolate intermediate via increased electron delocalization.
Voet, Voet & Pratt 2013 Fig. 15.5

22. Stage 2 of Glycolysis End of “stage I ” in Voet, Voet & Pratt
Berg, Tymoczko & Stryer, 2002 Chap 16.

23. Isomerization of Dihdroxyacetone phosphate
G=7.5kJ/mol
Glycolysis Step 5
Berg, Tymoczko & Stryer, 2002 Fig. 16.3

24. Isomerization of DHAP with Carbon #s
Lehninger 2000 Fig 15.4

25. Triose Phosphate Enzyme Mechanism
Cunningham 1978, p343

26. Triose Phosphate Isomerase Rx Proposed Mechanism
Glycolysis Step 5
Voet & Voet Biochemistry 1995 Fig.16.10

27. Catalytic Mechanism of Triose Phosphate Isomerase
Berg, Tymoczko & Stryer, 2012 Fig. 16.5

28. Avoiding Methyl Glyoxal by Triose Phosphate Isomerase
Berg, Tymoczko & Stryer, 2012 Chap 16 p. 460

29. Stage 3 Glycolysis Overview
Berg, Tymoczko & Stryer, Chap. 16 p.461
Voet, Voet & Pratt, 2013 Fig

30. Stage 3 of Glycolysis
Berg, Tymoczko & Stryer, 2002 Fig. 16.X

31. Conversion (Oxidation) of GAP into 1,3-BPG
G= 6.3 kJ/mol
Glycolysis Step 6
Berg, Tymoczko & Stryer, 2012 Chap.. 16 p. 461

32. Conversion of GAP into 1,3-BPG
Two steps involved: oxidation of aldehyde & joining of carboxylic acid with orthophosphate
G= 6.3 kJ/mol
Glycolysis Step 6
Berg, Tymoczko & Stryer, 2012 Chap. 16 p. 461

33. Glyceraldehyde-3-phosphate Dehydrogenase Mechanism
Enzyme active site
Glycolysis Step 6
Voet, Voet &Pratt 2013 Fig. 15.9

34. Glyceraldehyde Oxidation Free Energy Profile
Berg, Tymoczko & Stryer, 2012 Fig. 16.6
Berg, Tymoczko & Stryer, 2012 Fig. 16.6

35. Phosphoglycerate Kinase
G= kJ/mol
Glycolysis Step 7
Berg, Tymoczko & Stryer, 2012 Chap. 16 p. 463

36. Phosphoglycerate Kinase Reaction
Mechanism
Reaction
Glycolysis Step7
Voet & Voet Biochemistry p. 499

37. SUBSTRATE-LEVEL PHOSPHORYLATION
IMPORTANT: This refers to the formation of ATP from a high phosphoryl transfer potential substrate.
1,3-bisphosphoglycerate (1,3-BPG) in the phosphoglycerate kinase reaction of glycolysis is such an example.

38. Rearrangement of 3-phosphoglycerate
G= 4.4 kJ/mol
Glycolysis Step 8
Voet, Voet, & Pratt, 2013 Chap 15. p. 486

39. Phosphoglycerate Mutase Reaction Mechanism
Voet, Voet & Pratt 2008 Fig p500
Lehninger 2000 Fig 15.6

40. Phosphoglycerate Mutase Proposed Mechanism
Enzyme active site
Glycolysis Step 8
Voet & Voet Biochemistry 2013 Fig

41. Dehydration of 2-phosphoglycerate
G= 7.5 kJ/mol
Glycolysis Step 9
Voet, Voet, & Pratt 2012 Chap. 15 p. 487

42. Dephosphorylation of Phosphoenolpyruvate
G= kJ/mol
Glycolysis Step 10
Berg, Tymoczko & Stryer, 2002 Fig. 16.3;
2013 Chap 15 p. 465

43. Enzymes of Glycolysis Table
Bhagavan 2001 Biochemistry Table 13.2

44. Channeling of Intermediates in Glycolysis

45. The Redox Balance in Glycolysis
NADH Regeneration
Berg, Tymoczko & Stryer, 2012 Chap. 16 p. 466

46. Alcoholic Fermentation
Voet, Voet & Pratt Fig 15.18
Voet, Voet & Pratt 2013 Fig 15.16

47. Lactic Acid Fermentation
Berg, Tymoczko & Stryer, 2012 Chap. 16 p. 468

48. Redox Balance of NADH needed to Maintain Glycolysis
Berg, Tymoczko & Stryer, 2012 Fig

49. NAD+-Binding Domain of Dehydrogenases
Berg, Tymoczko & Stryer, 2012 Fig

50. Entry of other Hexoses into Glycolysis
Voet, Voet , & Pratt 2013 Fig 15.26

51. Galactose and Fructose Entry Points in Glycolysis
Berg, Tymoczko & Stryer, 2012 Fig

52. Fructose Metabolism
Voet, Voet & Pratt 2013 Fig 15.27

53. Galactose Metabolism
Voet, Voet & Pratt 2013 Fig 15.28

54. Feeder Pathways: Entry of Glycogen, Starch, Disaccharides and hexoses into preparatory stage of Glycolysis
Lehninger 2000 Fig 15.11

55. Control of the Glycolytic Pathway
The metabolic flux through the glycolytic pathway must be adjusted to respond to internal and extracellular conditions.
IMPORTANT - Two major cellular needs regulate the rate of glucose conversion into pyruvate:
1) The production of ATP.
2) The production of building blocks for synthetic reactions.
In metabolic pathways, enzymes catalyzing essentially irreversible reactions are potential sites for control.
These enzymes are regulated by allosteric effectors that reversibly bind to the enzyme or by covalent modification (meaning? E.g. phosphorylation).
These enzymes are also subject to regulation by transcription in response to metabolic loads (demands).

56. Regulation of Flux Through a Multistep Pathway
Lehninger 2000 Fig 15.16

57. Cumulative standard and actual free energy changes for the reactions of glycolysis
Horton et al 2012 Fig 11.12
Voet , Voet, & Pratt Table 15.1

58. Phosphofructokinase Control
For mammals, phosphofructokinase is the most important control element in the glycolytic pathway.
Voet, Voet & Pratt 2013 Fig 15.23
Berg, Tymoczko, & Stryer 2012 Fig 16.16

59. Phosphofructokinase Control II
Effect of F-2,6-BP and ATP
Berg, Tymoczko & Stryer, 2012 Fig

60. Glucagon Signal Pathway
Berg, Tymoczko & Stryer, 2012 Fig

61. Glycogen Phosphorylase of Liver as a Glucose Sensor
Lehninger 2000 Fig 15.19

62. Phosphofructokinase Control Summary of Regulatory Factors Affecting PFK
Lehninger 2000 Fig 15.18

63. Hexokinase Control
Hexokinase is inhibited by Glucose –6-P (its product). Indicates that the cell has sufficient energy supply. This will leave glucose in the blood.
Special case of liver: glucokinase (an isozyme) not inhibited by glucose-6-P. Has a 50-fold LOWER affinity for glucose. Functions to provide glucose-6-P for glycogen synthesis. Lower affinity means that hexokinase (muscle, brain) has first call on available glucose.

64. Pyruvate Kinase Control
Pyruvate kinase controls the outflow from the glycolysis pathway. It is the third irreversible step. This final step yields ATP and pyruvate.
Several mammalian isozymes of tetramer enzyme:
L-form predominates in liver
M-form predominates in muscle and brain
Berg, Tymoczko & Stryer, 2012 Fig

65. End of Lectures