1. Chapter 18 Glycolysis Biochemistry by Reginald Garrett and Charles Grisham

2. 18.1 – What Are the Essential Features of Glycolysis? The Embden-Meyerhof (Warburg) Pathway Consists of two phases: 1.First phase converts glucose to two Glyceraldehyde-3-P –Energy investment phase –Consumes 2 molecules of ATP 2.Second phase produces two pyruvates –Energy generation phase –Produces 4 molecules of ATP Products are 2 pyruvate, 2 ATP and 2 NADH Essentially all cells carry out glycolysis Ten reactions - same in all cells - but rates differ Three possible fates for pyruvate

3. Figure 18.1 The glycolytic pathway.

4. Figure 18.2 Pyruvate produced in glycolysis can be utilized by cells in several ways. In animals, pyruvate is normally converted to acetyl- coenzyme A, which is then oxidized in the TCA cycle to produce CO2. When oxygen is limited, pyruvate can be converted to lactate. Alcoholic fermentation in yeast converts pyruvate to ethanol and CO2.

5. 18.2 – Why Are Coupled Reactions Important in Glycolysis? Coupled reactions convert some, but not all, of the metabolic energy of glucose into ATP Under cellular conditions, approximately 5% of the energy of glucose is released in glycolysis Glucose + 2 ADP + 2 Pi  2 lactate + 2 ATP + 2H + + 2 H 2 O  G 0 ’ = -122.6 kJ/mol 2 ADP + 2 Pi  2 ATP + 2 H 2 O  G 0 ’ = 61 kJ/mol

6. 18.3 – What Are the Chemical Principles and Features of the First Phase of Glycolysis? 1.Phosphorylation (Hexokinase) 2.Isomerization (Phosphoglucoisomerase) 3.Phosphorylation (Phosphofructokinase) 4.Cleavage (Aldolase) 5.Isomerization (Triose phosphate isomerase)

7. Rx 1: Hexokinase The first reaction - phosphorylation of glucose Hexokinase (or glucokinase in liver) This is a priming reaction - ATP is consumed here in order to get more later ATP makes the phosphorylation of glucose spontaneous Mg 2+ is required

9. Magnesium-ATP complex (MgATP 2- ) Mg 2+ Under cellular condition  G =  G o ’ + RT ln ( [G-6-P][ADP] / [Glu][ATP] )

10. The cellular advantages of phosphorylating glucose 1.Phosphorylation keeps the substrate in the cell 2.Keeps the intracellular concentration of glucose low, favoring diffusion of glucose into the cell 3.Makes it an important site for regulation Figure 18.4 Glucose-6-P cannot cross the plasma membrane.

11. Hexokinase 1st step in glycolysis;  G large, negative K m for glucose is 0.1 mM; cell has 4 mM glucose, so hexokinase is normally active Hexokinase is regulated --allosterically inhibited by (product) glucose-6-P -- but is not the most important site of regulation of glycolysis—Why? ( Figure 18.5 ) Can phosphorylate a variety of hexose sugars, including glucose, mannose, and fructose

12. Figure 18.5 Glucose-6-phosphate is the branch point for several metabolic pathways.

13. Figure 18.6 The (a) open and (b) closed states of yeast hexokinase. Binding of glucose (green) induces a conformation change that closes the active site, as predicted by Daniel Koshland. The induced fit model for enzymes is discussed on page 409 of the text.

14. Figure 18.7 (a) Mammalian hexokinase I contains an N- terminal domain (top) and a C-terminal domain (bottom) joined by a long α- helix. Each of these domains is similar in sequence and structure to yeast hexokinase. (b) Human glucokinase undergoes induced fit upon binding glucose (green).

15. Glucokinase Glucokinase (K m glucose = 10 mM) only turns on when cell is rich in glucose Is not product inhibited Is an inducible enzyme by insulin

16. Rx 2: Phosphoglucoisomerase Glucose-6-P (aldose) to Fructose-6-P (ketose) Why does this reaction occur –next step (phosphorylation at C-1) would be tough for hemiacetal -OH, but easy for primary -OH –isomerization activates C-3 for cleavage in aldolase reaction Ene-diol intermediate in this reaction

17. Phosphoglucoisomerase, with fructose-6-P (blue) bound.

18. Figure 18.8 The phosphoglucoisomerase mechanism involves opening of the pyranose ring (step 1), proton abstraction leading to enediol formation (step 2), and proton addition to the double bond, followed by ring closure (step 3)

19. Rx 3: Phosphofructokinase PFK is the committed step in glycolysis The second priming reaction of glycolysis Committed step and large, negative  G -- means PFK is highly regulated Fructose-6-P + Pi → Fructose-1,6-bisP  G o’ = 16.3 kJ/mol

20. Regulation of Phosphofructokinase ATP is a allosteric inhibitor –Has two distinct binding sites for ATP –A high-affinity substrate site and a low-affinity regulatory site AMP reverses the inhibition due to ATP –Raise dramatically when ATP decrease Citrate is also an allosteric inhibitor Fructose-2,6-bisphosphate is allosteric activator PFK increases activity when energy status is low PFK decreases activity when energy status is high

21. Phosphofructokinase with ADP (in orange) and fructose- 6-phosphate (in red). Figure 18.9 At high ATP, phosphofructokinase (PFK) behaves cooperatively and the activity plot is sigmoid.

22. Figure 18.10 Fructose- 2,6-bisphosphate activates phosphofructokinase, increasing the affinity of the enzyme for fructose-6-phosphate and restoring the hyperbolic dependence of enzyme activity on substrate concentration.

23. Figure 18.11 Fructose-2,6- bisphosphate decreases the inhibition of phosphofructokinase due to ATP.

24. Rx 4: Aldolase C 6 is cleaved to 2 C 3 s (DHAP, Gly-3-P) Fructose bisphosphate aldolase cleaves fructose-1,6-bisphosphate between the C-3 and C-4 carbons to yield two triose phosphates Dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G-3-P)

25. The aldolase reaction is unfavorable as written at standard state. The cellular ΔG, however, is close to zero. The aldolase reaction in glycolysis is merely the reverse of the aldol condensation well known to organic chemists.

26. Animal aldolases are Class I aldolases Class I aldolases form covalent Schiff base intermediate between substrate and active site lysine

27. Class II aldolase are produced mainly in bacteria and fungi

28. Rx 5: Triose Phosphate Isomerase DHAP is converted to G3-P An ene-diol mechanism Active site Glu acts as general base Triose phosphate isomerase is a near-perfect enzyme

29. Figure 18.13 A reaction mechanism for triose phosphate isomerase. In the yeast enzyme, the catalytic residue is Glu 165. Triose phosphate isomerase with substrate analog 2- phosphoglycerate shown in cyan.

30. 18.4 – What Are the Chemical Principles and Features of the Second Phase of Glycolysis? Metabolic energy produces 4 ATP Net ATP yield for glycolysis is two ATP Second phase involves two very high energy phosphate intermediates –1,3 BPG –Phosphoenolpyruvate

32. 1.Oxidation and Phosphorylation (Glyceraldehyde-3-P Dehydrogenase) 2.Substrate-level Phosphorylation (phosphoglycerate kinase) 3.Isomerization (Phosphoglycerate isomerase) 4.Dehydration (Enolase) 5.Substrate-level Phosphorylation (pyruvate kinase)

33. Rx 6: Glyceraldehyde-3-Phosphate Dehydrogenase G-3-P is oxidized to 1,3-BPG Energy yield from converting an aldehyde to a carboxylic acid is used to make 1,3-BPG and NADH

34. G3PDH (or GAPDH) G-3-P is oxidized to 1,3-BPG Oxidation (aldehyde to carboxylic acid) and phosphorylation Mechanism involves covalent catalysis and a nicotinamide coenzyme This enzyme reaction is the site of action of arsenate – an anion analogous to phosphate

35. Figure 18.14 A mechanism for the glyceraldehyde-3- phosphate dehydrogenase reaction. Reaction of an enzyme sulfhydryl with G3P forms a thiohemiacetal, which loses a hydride to NAD+ to become a thioester. Phosphorolysis releases 1,3- bisphosphoglycerate. NADH

36. Rx 7: Phosphoglycerate Kinase ATP synthesis from a high-energy phosphate This is referred to as "substrate-level phosphorylation" Coupled reactions Glyceraldehyde-3-P + ADP + Pi + NAD + → 3-phosphoglycerate + ATP + NADH + H+  G o ’= -12.6 kJ/mol

37. ATP is synthesized by three major routes: 1.Substrate-level phosphorylation (Glycolysis, Citric acid cycle) 2.Oxidative phosphorylation (Driven by electron transport) 3.Photophosphorylation (Photosynthesis)

38. The open (a) and closed (b) forms of phosphoglycerate kinase. ATP (cyan), 3-phosphoglycerate (purple), and Mg 2+ (gold).

39. Figure 18.15 Formation and decomposition of 2,3-bisphosphoglycerate. 2,3-BPG (for hemoglobin) is made by circumventing the PGK reaction –Bisphosphoglycerate mutase –Erythrocytes contain 4-5 mM 2,3-BPG

40. Figure 18.16 The mutase that forms 2,3-BPG from 1,3-BPG requires 3-phosphoglycerate. The reaction is actually an intermolecular phosphoryl transfer from C-1 of 1,3-BPG to C-2 of 3-phosphoglycerate.

41. Rx 8: Phosphoglycerate Mutase Phosphoryl group from C-3 to C-2 Repositions the phosphate Mutase: catalyzes migration of a functional group within a substrate

42. Rx 8: Phosphoglycerate Mutase Phosphoenzyme intermediates A bit of 2,3-BPG is required as a cofactor Figure 18.17 A mechanism for the phosphoglycerate mutase reaction in rabbit muscle and in yeast.

43. The catalytic His183 at the active site of E. coli phosphoglycerate mutase

44. Rx 9: Enolase 2-PG to PEP Make a high-energy phosphate product Dehydration The enolase reaction creates a high-energy phosphate in preparation for ATP synthesis in step 10 of glycolysis.

45. Figure 18.18 The yeast enolase dimer is asymmetric. The active site of one subunit (a) contains 2-phosphoglycerate, the enolase substrate. The other subunit (b) binds phosphoenol- pyruvate, the product of the enolase reaction. Mg 2+ (blue); Li + (purple); water (yellow), and His 159 are shown.

46. Rx 10: Pyruvate Kinase PEP to Pyruvate makes ATP Substrate-level phosphorylation Another key control point for glycolysis Enol-keto tautomer The pyruvate kinase reaction converts PEP to pyruvate, driving synthesis of ATP. The two ATP produced here from one glucose are the “payoff” of glycolysis.

47. The structure of the pyruvate kinase tetramer is sensitive to bound ligands. Shown are the inactive E. coli enzyme in the absence of ligands (left), and the active form of the yeast dimer (right), with fructose-1,6-bisphosphate (an allosteric regulator, blue), substrate analog (red), and K + (gold).

48. Figure 18.19 The conversion of phosphoenolpyruvate (PEP) to pyruvate may be viewed as involving two steps: phosphoryl transfer, followed by an enol-keto tautomerization. The tautomerization is spontaneous and accounts for much of the free energy change for PEP hydrolysis.

49. Rx 10: Pyruvate Kinase Large, negative  G -- regulation Allosterically activated by AMP, F-1,6-bisP Allosterically inhibited by ATP,acetyl-CoA, and alanine Liver pyruvate kinase is regulated by covalent modification, responsive to hormonally-regulated phosphorylation in the liver (glucagon)- the phosphorylated form of the enzyme is less active.

50. Figure 18.20 A mechanism for the pyruvate kinase reaction, based on NMR and EPR studies by Albert Mildvan and colleagues. Phosphoryl transfer from PEP to ADP occurs in four steps.

51. 18.5 – What Are the Metabolic Fates of NADH and Pyruvate Produced in Glycolysis? Aerobic or anaerobic ? NADH must be recycled to NAD + –If O 2 is available, NADH is re-oxidized in the electron transport pathway, making ATP in oxidative phosphorylation (chapter 20) –In anaerobic conditions, NADH is re-oxidized by lactate dehydrogenase (LDH), providing additional NAD + for more glycolysis

52. Figure 18.21 (a) Pyruvate reduction to ethanol in yeast provides a means for regenerating NAD+ consumed in the glyceraldehyde-3-P dehydrogenase reaction. (b) In oxygen-depleted muscle, NAD+ is regenerated in the lactate dehydrogenase reaction.

53. Pyruvate also has two possible fates: –aerobic: into citric acid cycle (chapter 19) where it is oxidized to CO 2 with the production of additional NADH (and FADH 2 ) –anaerobic: (fermentation) 1.In yeast: reduced to ethanol Pyruvate decarboxylase (TPP) Alcohol dehydrogenase (Reoxidized NADH to NAD +) 2.In animals: reduced to lactate Lactate dehydrogenase (Reoxidized NADH to NAD +)

54. 18.6 – How Do Cells Regulate Glycolysis? The elegant evidence of regulation (See Figure 18.22) Standard state  G values are variously positive and negative   G in cells is revealing: –Most values near zero (reactions 2 and 4-9) –3 of 10 Reactions have large, negative  G Large negative  G Reactions are sites of regulation 1.Hexokinase 2.Phosphofructokinase 3.Pyruvate kinase

56. Overview of the regulation of glycolysis.

57. 18.7 – Are Substrates Other Than Glucose Used in Glycolysis? Fructose, mannose and galactose Fructose and mannose are routed into glycolysis by fairly conventional means. Figure 18.23 Mannose, galactose, fructose, and other simple metabolites can enter the glycolytic pathway.

58.  Fructose 1.In liver Fructokinase Fructose + ATP  fructose-1-phosphate + ADP + H + Fructoase-1-phosphate aldolase fructose-1-phosphate  glyceraldehyde + DHAP Triose kinase glyceraldehyde  glyceraldehyde-3-phosphate 2.In kidney and muscle Hexokinase Fructose + ATP  fructose-6-phosphate + ADP + H +

59.  Mannose 1.Hexokinase mannose + ATP  mannose-6-phosphate + ADP + H + 2.Phosphomannoisomerase mannose-6-phosphate  fructose-6-phosphate  Galactose is more interesting - the Leloir pathway "converts" galactose to glucose 1.Galactokinase Galactose + ATP  galactose-1-phosphate + ADP + H + 2.Galactose-1-phosphate uridylyltransferase 3.Phosphoglucomutase 4.UDP-galactose-4-epimerase

60. Figure 18.24 Galactose metabolism via the Leloir pathway.

61. Figure 18.25 The galactose-1-phosphate uridylyltransferase reaction involves a “ping-pong” kinetic mechanism.

62. Figure 18.26 The UDP-glucose pyrophosphorylase reaction. Galactosemia ─ defects in galactose-1-P uridylyltransferase ─ causes cataracts and permanent neurological disorders ─ in adults, UDP-glucose pyrophosphorylase can accept galactose

63. Glycerol can also enter glycolysis –glycerol is produced by the decomposition of triacylglycerols (chapter 23) –Converted to glycerol-3-phosphate by the action of glycerol kinase –Then oxidized to DHAP by the action of glycerol phosphate dehydrogenase –NAD + as the required coenzyme

64. 18.8 How Do Cells Respond to Hypoxic Stress? Glycolysis is an anaerobic pathway—it does not require oxygen The TCA (tricarboxylic acid) cycle is aerobic When oxygen is abundant, cells prefer to combine these pathways in aerobic metabolism When oxygen is limiting, cells adapt to carry out more glycolysis Hypoxia (oxygen limitation) causes changes in gene expression that increases –Angiogenesis (the growth of new blood vessels) –Synthesis of red blood cells –levels of some glycolytic enzymes

65. Hypoxic stress –A trigger for this is a DNA binding protein called hypoxia inducible factor (HIF) –HIF is regulated at high oxygen levels by hydroxylase factor-inhibiting HIF (FIH-1) Figure 18.27 FIH (green) bound to HIF. Hydroxylation of HIF-1α Asn 803 by FIH in the presence of oxygen inhibits the transcription activity of HIF-1α.

66. HIF consists of two subunits: a ubiquitous HIF-1β subunit and a hypoxia-responsive HIF-1α subunit In response to hypoxia, inactivation of the prolyl hydroxylases (PHDs) allows –HIF-1α stabilization –Dimerization with HIF-1β –Binding of the dimer to the hypoxia responsive element (HRE) of HIF target genes –Activating transcription of these genes VHL is the von Hippel Lindau subunit of the ubiquitin E3 ligase that targets proteins for proteasome degradation

67. Figure 18.28