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Chapter 18

Chapter 18. Glycolysis Biochemistry by Reginald Garrett and Charles Grisham. 18.1 – What Are the Essential Features of Glycolysis?. The Embden-Meyerhof (Warburg) Pathway: Glycolysis Consists of two phases: First phase converts glucose to two Glycer aldehyde -3-P

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Chapter 18

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  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: Glycolysis • Consists of two phases: • First phase converts glucose to two Glyceraldehyde-3-P • Energy investment phase • Consumes 2 molecules of ATP • 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

  3. Figure 18.1The 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 • The free energy change for the conversion of glucose to two lactates is -1863.6 Glucose  2 lactate + 2H+DG0’ = -183.6 kJ/mol • The production of two molecules of ATP in glycolysis is an energy-required process 2 ADP + 2 Pi  2 ATP + 2 H2O DG0’ = 61 kJ/mol

  6. Glycolysis couples these two reactions Glucose  2 lactate + 2H+DG0’ = -183.6 kJ/mol 2 ADP + 2 Pi  2 ATP + 2 H2O DG0’ = 61 kJ/mol Glucose + 2 ADP + 2 Pi  2 lactate + 2 ATP + 2H+ + 2 H2O DG0’ = -122.6 kJ/mol • More than enough free energy is available in the conversion of glucose into lactate to drive the synthesis of two molecules of ATP

  7. 18.3 – What Are the Chemical Principles and Features of the First Phase of Glycolysis?

  8. Under cellular conditionDG = DGo’ + RT ln ( [G-6-P][ADP] / [Glu][ATP] )

  9. Phase 1 • Phosphorylation (Hexokinase) • Isomerization (Phosphoglucoisomerase) • Phosphorylation (Phosphofructokinase) • Cleavage (Aldolase) • Isomerization (Triose phosphate isomerase)

  10. Reaction 1: Hexokinase 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 • Mg2+ is required Mg2+

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

  12. Hexokinase1st step in glycolysis; G large, negative • Km 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

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

  14. Glucokinase • The isozymes of hexokinase • Hexokinase I in brain • Hexokinase I (75%, 0.03mM) and II (25%, 0.3mM) in muscle • Glucokinase in liver and pancreas • Glucokinase (Kmglucose = 10 mM) only turns on when cell is rich in glucose • Is not product inhibited • Is an inducible enzyme by insulin

  15. Induced fit model (fig 13.24) Figure 18.7 (a) Mammalian hexokinase I Figure 18.6 The (a) open and (b) closed states of yeast hexokinase

  16. Reaction 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 • Phosphoglucose isomerase or glucose phosphate isomerase • 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. Reaction 3: Phosphofructokinase PFK is the committed step in glycolysis • The second priming reaction of glycolysis • Committed step and large, negative DG -- means PFK is highly regulated Fructose-6-P + Pi → Fructose-1,6-bisP DGo’= 16.3 kJ/mol

  20. Regulation of Phosphofructokinase • ATP also 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. 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. Reaction 4: Fructose Bisphosphate Aldolase C6 is cleaved to2 C3s (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. Reaction 5: Triose Phosphate Isomerase • Only G-3-P goes directly into the second phase, DHAP must be converted to G-3-P • Triose phosphate isomerase • An ene-diol mechanism • Active site Glu acts as general base • is a near-perfect enzyme (Table 13.5)

  29. Figure 18.13 A reaction mechanism for triose phosphate isomerase. In the yeast enzyme, the catalytic residue is Glu165.

  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 • Substrate-level phosphorylation

  31. Phase 2 Oxidation and Phosphorylation (Glyceraldehyde-3-P Dehydrogenase) Substrate-level Phosphorylation (phosphoglycerate kinase) Isomerization (Phosphoglycerate isomerase) Dehydration (Enolase) Substrate-level Phosphorylation (pyruvate kinase)

  32. Reaction 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 • Oxidation (aldehyde to carboxylic acid) and phosphorylation

  33. G3PDH (or GAPDH) • Mechanism involves covalent catalysis and a nicotinamide coenzyme

  34. Reaction 7: Phosphoglycerate Kinase ATP synthesis from a high-energy phosphate • This is referred to as "substrate-level phosphorylation" • Coupled reactions; 6th and 7th reactions Glyceraldehyde-3-P + ADP + Pi + NAD+→ 3-phosphoglycerate + ATP + NADH + H+ DGo’= -12.6 kJ/mol

  35. ATP is synthesized by three major routes: • Substrate-levelphosphorylation(Glycolysis, Citric acid cycle) • Oxidative phosphorylation(Driven by electron transport) • Photophosphorylation (Photosynthesis)

  36. 2,3-BPG (for hemoglobin) is made by circumventing the PGK reaction • Bisphosphoglycerate mutase • Erythrocytes contain 4-5 mM 2,3-BPG Figure 18.16 The mutase that forms 2,3-BPG from 1,3-BPG requires 3-phosphoglycerate.

  37. Reaction 8: Phosphoglycerate Mutase • Repositions the phosphate • Mutase: catalyzes migration of a functional group within a substrate

  38. Phosphoenzyme intermediates • A bit of 2,3-BPG is required as a cofactor The catalytic His183 at the active site of E. coli phosphoglycerate mutase

  39. Reaction 9: Enolase • The formation of PEP from 2-PG • Dehydration • Make a high-energy phosphate in preparation for ATP synthesis in step 10 of glycolysis

  40. Reaction 10: Pyruvate Kinase • The pyruvate kinase reaction converts PEP to pyruvate, driving synthesis of ATP. • Substrate-level phosphorylation • Another key control point for glycolysis • Enol-keto tautomer .

  41. 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.

  42. Large, negative G -- regulation • Allosteric regulation • Activated by AMP, F-1,6-bisP • 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 more strongly inhibited by ATP and alanine. • Has a higher Km for PEP

  43. 18.5 – What Are the Metabolic Fates of NADH and Pyruvate Produced in Glycolysis? Aerobic or anaerobic? • NADH must be recycled to NAD+ • If O2 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

  44. 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.

  45. Pyruvate also has two possible fates: • aerobic: into citric acid cycle (chapter 19) where it is oxidized to CO2 with the production of additional NADH (and FADH2) • anaerobic: (fermentation) • In yeast: reduced to ethanol • Pyruvate decarboxylase (TPP) • Alcohol dehydrogenase (Reoxidized NADH to NAD+) • In animals: reduced to lactate • Lactate dehydrogenase (Reoxidized NADH to NAD+)

  46. 18.6 – How Do Cells Regulate Glycolysis? The elegant evidence of regulation (See Figure 18.22) • Standard state G values are variously positiveand 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 • Hexokinase • Phosphofructokinase • Pyruvate kinase

  47. Overview of the regulation of glycolysis.

  48. 18.7 – Are Substrates Other Than Glucose Used in Glycolysis? Sugars other than glucose can be glycolytic substrates • Fructose and mannose are routed into glycolysis by fairly conventional means.

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