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Glycolysis

Glycolysis. Chapter 15. Definitions, notes. Sequence of 10 rxns Converts glu  pyruvate Some ATP Divided – 5 “preparatory”, 5 “payoff” Glycolytic intermediates 6C – deriv’s of glu or fru 3C – deriv’s of dihydroxyacetone, glyceraldehye. Fig.15-2. Fig.15-2. Definitions, notes -- cont’d.

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Glycolysis

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  1. Glycolysis Chapter 15

  2. Definitions, notes • Sequence of 10 rxns • Converts glu  pyruvate • Some ATP • Divided – 5 “preparatory”, 5 “payoff” • Glycolytic intermediates • 6C – deriv’s of glu or fru • 3C – deriv’s of dihydroxyacetone, glyceraldehye

  3. Fig.15-2

  4. Fig.15-2

  5. Definitions, notes -- cont’d • All intermediates phosph’d as esters or anhydrides • Net neg charge • Raises free energy of reactants • Enz active sites specific for ADP/ATP/intermediate complexes w/ Mg+2

  6. Definitions, notes -- cont’d • 5 types of rxns • phosphoryl transfer • phosphoryl shift • isomerization • dehydration • aldol cleavage • In cell cytosol

  7. Definitions, notes -- cont’d • Overall • Glu + 2 NAD+ + 2 ADP + 2 Pi  2 Pyruvate + 2 NADH + 2 H+ + 2 ATP + 2 H2O • D Go’ entire rxn = -85 kJ/mole • Pyruvate product (if aerobic cond’s)  TCA  e- transport/ox’ve phosph’n  ATP gen’d (15-3) • From glycolysis  ATP yields ~2800 kJ/mole • No O2 = anaerobic metab = diff pathway = diff energy

  8. Regulation of Glycolysis • 3 Cell mechanisms • 1. Regulation of enz catalytic activity • Allosteric control • Enz’s have sev subunits • Modulators bind @ binding site • Often regulatory subunit • Causes conform’l change •  conform’l change @ catalytic subunit •  Stimulation or inhibition

  9. Regulation of Glycolysis -- cont’d • 1. Reg’n enz activity -- cont’d • (Reversible) covalent mod’n • Enz’s have other enz’s assoc’d • Other enz’s catalyze covalent binding of funct’l grp to reg enz (or removal of funct’l grp) •  Stimulation or inhibition

  10. Regulation of Glycolysis -- cont’d • 2. Regulation of concent of enz’s in cell • Rates of enz synth, degrad’n impt • When incr’d substrate (chronic), •  Incr’d transcr’n genes coding •  Incr’d concent enz’s impt to pathway

  11. Regulation of Glycolysis -- cont’d • 3. Regulation of flux of substrates • Cell can allow more substrate into cell •  Incr’d activity of pathway •  Incr’d prod’n • Hormones impt

  12. Glu  Glu-6-PO4

  13. Hexokinase • Phosphoryl transfer • Hydrol ATP  ADP + Pi • Cofactor Mg+2 • Reversible? • Induced fit (8-21) • Isozymes in mammals

  14. Fig.8-21

  15. Glu-6-PO4 Fru-6-PO4

  16. Phosphohexose isomerase • Aldose  ketose • Mg+2 cofactor • Reversible

  17. Fru-6-PO4 Fru-1,6-Bisphosphate

  18. Phosphofructokinase-1 (PFK-1) • Phosphoryl transfer • Hydrol ATP • Mg+2 cofactor • Reversible?

  19. PFK-1 -- cont’d • Regulatory enz • Commits to glycolysis • Impt to regulation of pathway • Sev binding sites for modulators (15-18c)

  20. PFK-1 Modulators • 1. Adenine nucleotides •  PFK-1 activity (inhib’n) when  [ATP] or other fuels • ATP binds allosteric site •  affinity for fru-6-PO4 •  activity (stim’d) when  [ADP]/[AMP] or  [ATP] • ADP/AMP bind allosterically •  Stm’n PFK-1 •  More ATP overall in cell

  21. Fig.15-18a

  22. PFK-1 Modulators -- cont’d • 1. Adenine nucleotides -- cont’d • Note: If  [ATP] in cell, ATP acts as feedback inhibitor to decr its further synth • As  ATP synth, and ATP used,  [ADP], [AMP] • Signals cell to restart ATP syth, so ADP, AMP act as “feedback stimulators” to incr ATP synth again

  23. Fig.15-18b

  24. PFK-1 Modulators -- cont’d • 2. Citrate • Intermed formed in Kreb’s cycle •  PFK-1 activity when  [citrate] • Citrate binds allosteric site • Usually concurrent w/ ATP modulation • So feedback inhib’n

  25. PFK-1 Modulators -- cont’d • 3. Fru-2,6-Bisphosphate (p.554) • In liver •  PFK-1 activity when  [Fru-2,6-bisphosphate] • Binds allosteric site •  affinity of PFK-1 for fru-6-PO4 • Acts as allosteric stimulator of PFK-1 • When Fru-2,6-bis… present, glycolysis encouraged

  26. PFK-1 Modulators -- cont’d • 3. Fru-2,6-Bisphosphate -- cont’d • Helps balance glu used in cell w/ glu generated (gluconeogenesis) • Works through hormone glucagon • Rel’d from pancreas • When  [blood glu] • Glucagon  cell membr receptor  adenylate cyclase activation  cAMP prod’n  stim’n cell prot kinases

  27. PFK-1 Modulators -- cont’d • 3. Fru-2,6-Bisphosphate -- cont’d • In liver, stim’n cell prot kinases  fru-2,6-bisphosphate (glycolysis [glu metab] discouraged) • So PFK-1 NOT stim’d to metab glu • Rather, this tells cell to  glu prod’n

  28. Fru-1,6-Bisphosphate  Dihydroxyacetone PO4 + Glyceraldehyde-3-PO4

  29. Aldolase • Reverse aldol condensation • Reversible? • Proceeds readily as 2P’s immediately  subsequent rxns • Have committed to pathway • Where was commitment?

  30. Dihydroxyacetone PO4 Glyceraldehyde-3-PO4

  31. Triose phosphate isomerase • Reversible? • Enediol intermediate • Glu 165 –COOH, His 95 –H participate • Lys –NH3 “holds” –PO4 • kcat/KM shows kinetically perfect enzyme activity

  32. Priming phase ended here • 6C glu  2 3C phosph’d cmpds • More red’d  more ox’d • Consumed 2 ATP from cell • Cell energy “invested” • Will yield more energy for cell at end of pathway

  33. Fig.15-4 • REMEMBER: for each future step, the cell has twice as many molecules as started out (each 1 glu  2 glyc-3-PO4)

  34. Glyceraldehyde-3-PO4 1,3-Bisphosphoglycerate

  35. Glyceraldehyde-3-PO4 Dehydrogenase • Where did you hear about dehydrogenases before? • HINT: 1st step leading to ATP prod'n through e- transport • Aldehyde now  carboxylic acid anhydride w/ PO4 • High D G of hydrolysis (-49.3 kJ/mole)

  36. Rxn Mechanism: Glyc-3-PO4 DeHase (15-5)

  37. Cys in enz active site forms thiohemiacetal w/ glyc-3-PO4 aldehyde grp • So S cov'ly bound to E in active site • 1 H+ given off to sol'n

  38. Fig.15-5 • Note: iodoacetate is inhibitor by cov'ly binding cys-SH

  39. 1 :H- reduces NAD+ • Cofactor of enz • Now red'd  NADH

  40.  thioester @ active site • Energy-rich intermediate • Note electrophilicity/ dipole moment

  41. 2nd NAD+ accepts :H- from cofactor •  NADH avail to transport e- to mitoch for e- transport/ox'v phosph'n/ATP synth • Ox'd cofactor now regen'd

  42. Thioester is good target for phosphate attack • Energy rel'd w/ cleavage of thioester by phosphate •  Acyl phosphate product + enz regen'd

  43. 1,3-Bisphosphoglycerate + ADP  3-Phosphoglycerate + ATP

  44. Phosphoglycerate Kinase • Requires Mg+2 • Substrate-level phosphorylation • In cytosol • Ox've phosph'n in mitoch • Coupled w/ preceding rxn to allow overall neg D G • Book notes E inc'd into ATP "from" ox'n aldehyde (step 6)  carboxylic acid (step 7)

  45. 3-Phosphoglycerate  2-Phosphoglycerate

  46. Phosphoglycerate Mutase • Reversible; ex of cov'ly mod'd enz • Enz has impt His @ active site • Stim'd w/ phosph'n • Must be "primed" by:

  47. Phosphoglycerate Mutase Mechanism • Assoc'd kinase phosphorylates S (3-phospho glycerate) of enz • From ATP •  2,3-Bisphospho glycerate

  48. 2,3-Bisphospho glycerate phosphorylates enz @ active site His  Phosph'd enz (stimulated) + 3-Phospho- glycerate regen'd

  49. 3-Phospho glycerate enters active site • Phosph'd  2,3-bisPO4glycerate • Catalyzed by phosph'd enz •  Inactive (dephosph'd) enz regenerated

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