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Glycolysis II 11/05/09

Glycolysis II 11/05/09. Front half of glycolysis. Aldolase with a Tyr residue acting as a proton donor / acceptor. Aldolase with an Asp residue acting as a proton donor / acceptor (current text book). Triosephosphate isomerase. DHAP GAP .

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Glycolysis II 11/05/09

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  1. Glycolysis II11/05/09

  2. Front half of glycolysis

  3. Aldolase with a Tyr residue acting as a proton donor / acceptor

  4. Aldolase with an Asp residue acting as a proton donor / acceptor (current text book)

  5. Triosephosphate isomerase DHAP GAP TIM is a perfect enzyme which its rate is diffusion controlled. A rapid equilibrium allows GAP to be used and DHAP to replace the used GAP.

  6. TIM has an enediol intermediate GAP enediol DHAP Transition state analogues Phosphoglycohydroxamate (A) and 2-phosphoglycolate (B) bind to TIM 155 and 100 times stronger than GAP of DHAP B. A.

  7. TIM has an extended “low barrier” hydrogen bond transition state Hydrogen bonds have unusually strong interactions and have lead to pK of Glu 165 to shift from 4.1 to 6.5 and the pK of

  8. Geometry of the enediol intermediate prevents formation of methyl glyoxal Orbital symmetry prevents double bond formation needed for methyl glyoxal

  9. The second half of glycolysis

  10. Glyceraldehyde-3-phosphate dehydrogenase The first high-energy intermediate +NAD+ + Pi + NADH Uses inorganic phosphate to create 1,3 bisphosphoglycerate

  11. Reactions used to elucidate GAPDH’s mechanism

  12. Mechanistic steps for GAPDH 1. GAP binds to enzyme. 2. The nucleophile SH attacks aldehyde to make a thiohemiacetal. 3. Thiohemiacetal undergoes oxidation to an acyl thioester by a direct transfer of electrons to NAD+ to form NADH. 4. NADH comes off and NAD+ comes on. 5. Thioester undergoes nucleophilic attack by Pi to form 1,3 BPG. The acid anhydride of phosphate in a high energy phosphate intermediate

  13. Arsenate uncouples phosphate formation 3PG GAP DH + +

  14. Phosphoglycerate Kinase: First ATP generation step + ADP + ATP 3 PG 1,3 BPG

  15. GAP + Pi + NAD+ 1,3 -BPG + NADH + 6.7 kJ• mol-1 1,3 BPG + ADP 3PG + ATP -18.8 kJ• mol-1 GAP+Pi+NAD+ +ADP 3PG+NADH+ATP -12.1 kJ•mol-1

  16. Phosphoglycerate mutase 2PG 3 PG 2,3 BPG

  17. Phosphoglycerate mutase requires a phosphorylated form of the enzyme to be active. Only 2,3 BPG can phosphorylate the unphosphorylated enzyme. Phospho Histidine residue

  18. Glycolysis influences oxygen transport Oxygen saturation curves in erythrocytes

  19. Enolase generation of a second “high energy” intermediate + H2O 2 PhosphoglyceratePhosphoenol pyruvate

  20. Pyruvate kinase: Second ATP generation step

  21. Next LectureTuesday 11/10/09 Glycolysis III

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