Glycolysis

1. Glycolysis Chapter 14

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

5. 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. 5 types of rxns • phosphoryl transfer • phosphoryl shift • isomerization • dehydration • aldol cleavage • In cell cytosol

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

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

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

11. 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

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

14. Glu  Glu-6-PO4

15. Hexokinase • Phosphoryl transfer from ATP • Type of transferase • Hydrol ATP  ADP + Pi • Other hexose substrates • Cofactor Mg+2 • Reversible? • Induced fit w/ glu binding (Chpt 6) • Isozymes in mammals

17. Glu-6-PO4 Fru-6-PO4

18. Phosphohexose Isomerase • Aldose  ketose • Mg+2 cofactor • Reversible • Mechanism through enediol intermediate

19. His plays role in steps 1,4 B:=Glu

20. Fru-6-PO4Fru-1,6-Bisphosphate

21. Phosphofructokinase-1 (PFK-1) • Phosphoryl transfer w/ hydrol ATP • Mg+2 cofactor • Reversible? • Regulatory enz • Commits to glycolysis • Impt to regulation of pathway • Sev binding sites for modulators (Chpt 15)

22. 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

23. Blue=ADP Yellow=fru-1,6-bisphosphate

24. 1. Adenine nucleotides -- cont’d • Note: If  [ATP] in cell, ATP  feedback inhib to decr 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

26. 1. Adenine nucleotides -- cont’d • Also impt to balancing glycolysis w/ gluconeogenesis (“making new glucose”) • Uses sev enz’s impt in glycolysis (reversed) • BUT other, diff enz’s allow separation of pathways, regulation of 2 (so no “futile cycles”) • Gluconeogenesis alternative to PFK-1 cat’d by fructose-1,6-bisphosphatase (FBPase-1) • AMP stim’s PFK-1 (when more ATP needed by cell, much glu avail), BUT inhib’s FBPase-1 (when cell needs more glu, not enough avail to make more ATP)

27. Chpt 15

28. 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

29. 3. Fru-2,6-Bisphosphate • 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, gluconeogenesis discouraged

30. 2 1

31. Chpt 15

32. 3. Fru-2,6-Bisphosphate -- cont’d • Helps balance glu used in cell w/ glu generated (gluconeogenesis) • Impt to maintaining [blood glu] • Works through hormone glucagon • If not enough blood glu •  stim’n ad cyclase/cAMP/prot kinase pathway if gluconeogenesis nec because not enough nutrient glu avail to maintain sufficient [blood glu]

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

34. Aldolase • Reverse aldol condensation • Schiff base form’n; enamine intermediate • Reversible? • Proceeds readily as 2P’s immediately  subsequent rxns • Have committed to pathway • Where was commitment?

36. Dihydroxyacetone PO4 Glyceraldehyde-3-PO4

37. Triose Phosphate Isomerase • Reversible? • Enediol intermediate (sim to phosphohexose isomerase mech) • Glu 165 –COOH, His 95 –H participate • Lys –NH3 “holds” –PO4 • kcat/KM shows kinetically perfect enzyme activity

38. Priming Phase Ends Here; Payoff Phase to Begin • 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 • REMEMBER: for each future step, cell has 2x the mol’s as began (each 1 glu  2 glyc-3-PO4)

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

42. 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)

43. Rxn Mechanism: Glyc-3-PO4 DeHase

44. Cys in enz active site forms thiohemiacetal w/ glyc-3-PO4 aldehyde grp • So S cov'ly bound to E in active site

45. 1 :H- reduces NAD+ • Cofactor of enz • Now NADH •  thioester @ active site • Energy-rich intermediate

46. 2nd NAD+ enters, accepts :H- from orig NAD cofactor •  NADH avail to transport e- to mitoch for e- transport/ox'v phosph'n/ATP synth • Ox'd cofactor regen'd • Pi enters • Thioester good target for phosphate attack • Energy rel'd w/ attack, cleavage of thioester by phosphate

47. Cleavage w/ phosph’n  prod released and active site regen’d

48. 1,3-Bisphosphoglycerate + ADP  3-Phosphoglycerate + ATP

49. 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)  carbox acid (step 7)

50. 3-Phosphoglycerate 2-Phosphoglycerate