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Chapter 19

Chapter 19. Glycolysis to accompany Biochemistry, 2/e by Reginald Garrett and Charles Grisham.

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Chapter 19

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  1. Chapter 19 Glycolysis to accompany Biochemistry, 2/e by Reginald Garrett and Charles Grisham All rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company, 6277 Sea Harbor Drive, Orlando, Florida 32887-6777

  2. Outline • 19.1 Overview of Glycolysis • 19.2 Coupled Reactions in Glycolysis • 19.3 First Phase of Glycolysis • 19.4 Second Phase of Glycolysis • 19.5 Metabolic Fates of NADH and Pyruvate • 19.6 Anaerobic Pathways for Pyruvate • 19.7 Energetic Elegance of Glycolysis • 19.8 Other Substrates in Glycolysis

  3. Overview of Glycolysis The Embden-Meyerhof (Warburg) Pathway • Essentially all cells carry out glycolysis • Ten reactions - same in all cells - but rates differ • Two phases: • First phase converts glucose to two G-3-P • Second phase produces two pyruvates • Products are pyruvate, ATP and NADH • Three possible fates for pyruvate

  4. First Phase of Glycolysis The first reaction - phosphorylation of glucose • Hexokinase or glucokinase • This is a priming reaction - ATP is consumed here in order to get more later • ATP makes the phosphorylation of glucose spontaneous • Be SURE you can interconvert Keq and standard state free energy change • Be SURE you can use Eq. 3.12 to generate far right column of Table 19.1

  5. Hexokinase1st step in glycolysis; G large, negative • Hexokinase (and glucokinase) act to phosphorylate glucose and keep it in the cell • Km for glucose is 0.1 mM; cell has 4 mm glucose • So hexokinase is normally active! • Glucokinase (Kmglucose = 10 mM) only turns on when cell is rich in glucose • Hexokinase is regulated - allosterically inhibited by (product) glucose-6-P - but is not the most important site of regulation of glycolysis - Why?

  6. Rx 2: Phosphoglucoisomerase Glucose-6-P to Fructose-6-P • 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 • Be able to write a mechanism!

  7. Rx 3: Phosphofructokinase PFK is the committed step in glycolysis! • The second priming reaction of glycolysis • Committed step and large, neg delta G - means PFK is highly regulated • ATP inhibits, AMP reverses inhibition • 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

  8. Rx 4: Aldolase C6 cleaves to 2 C3s (DHAP, Gly-3-P) • Animal aldolases are Class I aldolases • Class I aldolases form covalent Schiff base intermediate between substrate and active site lysine • Understand the evidence for Schiff base intermediate (box on page 622)

  9. Rx 5: Triose Phosphate Isomerase DHAP converted to Gly-3-P • An ene-diol mechanism (know it!) • Active site Glu acts as general base • Triose phosphate isomerase is a near-perfect enzyme - see Table 14.5

  10. Glycolysis - Second Phase 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

  11. Rx 6: Gly-3-Dehydrogenase Gly-3P 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 • Mechanism is one we saw in Chapter 16 (see Figure 16.10!) • Mechanism involves covalent catalysis and a nicotinamide coenzyme - know it

  12. Rx 7: Phosphoglycerate Kinase ATP synthesis from a high-energy phosphate • This is referred to as "substrate-level phosphorylation" • 2,3-BPG (for hemoglobin) is made by circumventing the PGK reaction

  13. Rx 8: Phosphoglycerate Mutase Phosphoryl group from C-3 to C-2 • Rationale for this enzyme - repositions the phosphate to make PEP • Note the phospho-histidine intermediates! • Zelda Rose showed that a bit of 2,3-BPG is required to phosphorylate His

  14. Rx 9: Enolase 2-P-Gly to PEP • Overall G is 1.8 kJ/mol • How can such a reaction create a PEP? • "Energy content" of 2-PG and PEP are similar • Enolase just rearranges to a form from which more energy can be released in hydrolysis

  15. Rx 10: Pyruvate Kinase PEP to Pyruvate makes ATP • These two ATP (from one glucose) can be viewed as the "payoff" of glycolysis • Large, negative G - regulation! • Allosterically activated by AMP, F-1,6-bisP • Allosterically inhibited by ATP and acetyl-CoA • Understand the keto-enol equilibrium of Py

  16. The Fate of NADH and PyruvateAerobic or anaerobic?? • NADH is energy - two possible fates: • If O2 is available, NADH is re-oxidized in the electron transport pathway, making ATP in oxidative phosphorylation • In anaerobic conditions, NADH is re-oxidized by lactate dehydrogenase (LDH), providing additional NAD+ for more glycolysis

  17. The Fate of NADH and PyAerobic or anaerobic?? • Pyruvate is also energy - two possible fates: • aerobic: citric acid cycle • anaerobic: LDH makes lactate

  18. Energetics of Glycolysis The elegant evidence of regulation! • See Figure 16.31 • Standard state G values are scattered: + and - • G in cells is revealing: • Most values near zero • 3 of 10 Rxns have large, negative  G • Large negative  G Rxns are sites of regulation!

  19. Other Substrates for Glycolysis Fructose, mannose and galactose • Fructose and mannose are routed into glycolysis by fairly conventional means. See Figure 18.32 • Galactose is more interesting - the Leloir pathway "converts" galactose to glucose - sort of.... • See Figure 16.33

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