Overview of Glycolysis

1. Overview of Glycolysis • The Embden-Meyerhof (Warburg) Pathway • Essentially all cells carry out glycolysis • Cellular location: cytosol • Ten reactions - same in all cells - but rates differ • Two phases: • 1st phase- investing phase: glucose →F-1,6-2P →2G-3-P • 2nd phase-harvesting phase: produces two pyruvates, ATPs and NADH • Three possible fates for pyruvate

2. Overview of Glycolysis

4. Glucose (6C) C-C-C-C-C-C 2ATP 2 ATP - used 0 ATP - produced 0 NADH - produced 2ADP + P C-C-C C-C-C Glyceraldehyde phosphate (2 - 3C) (G3P or GAP) Glycolysis • A. Energy Investment Phase:

5. ⑤

6. Glyceraldehyde phosphate (2 - 3C) (G3P or GAP) GAP GAP C-C-C C-C-C 4ADP + P 0 ATP - used 4 ATP - produced 2 NADH - produced 4ATP C-C-C C-C-C (PYR) (PYR) Pyruvate (2 - 3C) (PYR) Glycolysis • B. Energy Yielding Phase

8. Glycolysis

9. Overview of Glycolysis

10. You are not responsible for any structures Don’t panic! • All you have to memorize are the total reaction, three rate-limiting reactions, three inhibitors, two substrate-level phosphorylation steps and major regulation mechanisms.

11. 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

12. 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

15. A good illustration for the induced fit model

16. Induced fit: Binding of glucose to Hexokinase induces a large conformational change • This same change in conformation is observed for ALL kinases! It also accounts for the fact that water cannot be used for hydrolysis of ATP unless we fool the enzyme by using xylose instead of glucose.

17. 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 • A moonlighting protein-double as a kind of nerve growth factor

19. Rx 3: Phosphofructokinase • PFK is the committed step in glycolysis! • The second priming reaction of glycolysis • Committed step and large, -ΔG - means PFK is highly regulated

21. 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

22. 4 1 5 2 6 3

24. 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

27. 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

28. 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 involves covalent catalysis and a nicotinamide coenzyme - know it • Arsenate (AsO43-) can substitute for HPO42- in this rxn because it is very similar in structure and reactivity but resulting 1-arseno-3-phosphoglycerate is unstable and will spontaneously hydrolyze to 3-phosphoglycerateOxidation of substrate is uncoupled from ATP synthesis: arsenate is a potent poison! Glycolysis becomes futile - no usable energy is extracted from glucose

32. 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

33. Enzyme O- C=O C-O- CH2 Adenosine P P P Substrate ADP (PEP) O- C=O C=O CH2 Product (Pyruvate) Adenosine P P P ATP Substrate-Level Phosphorylation • ATP is formed when an enzyme transfers a phosphate groupfrom a substrate to ADP. Example: PEP to PYR

36. 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

39. 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

41. 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!

44. The Fate of NADH and PyruvateAerobic or anaerobic?? • NADH is energy - two possible fates: • If O2 is available, NADH enters into Mitochondria by two ways, where it 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

45. Fates of Pyruvate Potential energy = 2840 kJ/mol Go’= -146 kJ/mol Go’= -1160 kJ/mol Go’= -196 kJ/mol Go’= -235 kJ/mol

48. Energetics of Glycolysis • The elegant evidence of regulation! • 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!

50. Other Substrates for Glycolysis • Glycerol, Fructose, mannose and galactose • Gycerol is changed into DHAP • Fructose and mannose are routed into glycolysis by fairly conventional means. • Galactose is more interesting - the Leloir pathway "converts" galactose to glucose - sort of....