Glycolysis

1. Glycolysis Andy HowardBiochemistry Lectures, Spring 2019 28 March 2019

2. Glycoproteins Glycolysis Overview Reactions through TIM Final reactions to pyruvate Control What we’ll discuss Glycolysis

3. Glycoproteins (CF&M §16-5) • 1-30 carbohydrate moieties per protein • Proteins can be enzymes, hormones, structural proteins, transport proteins • Microheterogeneity:same protein, different sugar combinations • Eight sugars common in eukaryotes • PTM glycosylation much more common in eukaryotes than prokaryotes Glycolysis

4. Diversity in glycoproteins • Variety of sugar monomers •  or glycosidic linkages • Linkages always at C-1 (anomeric) on one sugar but can be C-2,3,4,6 on the other one • Up to 4 branches • But: not all the specific glycosyltransferases you would need to get all this diversity exist in any one organism Glycolysis

5. O-linked and N-linked oligosaccharides • Characteristic sugar moieties and attachment chemistries Glycolysis

6. O-linked oligosaccharides • GalNAc to Ser or Thr;often with gal or sialic acid on GalNAc • 5-hydroxylysines on collagen are joined to D-Gal • Some proteoglycans joined viaGal-Gal-Xyl-Ser • Single GlcNAc on ser or thr Glycolysis

7. Human ABO Blood Groups • Based on H antigen:cell-surface oligosaccharide that links to a small protein (or to a ceramide) • Invariant part of H antigen structure isFuc-Gal-GalNAc-Gal • Fucose is 6-deoxygalactose Glycolysis

8. ABO blood groups, continued • A antigen attaches another GalNAc to the first Gal • B antigen attaches another Gal to 1st Gal Glycolysis

9. Enzymology and Heritance • Fucosyltransferase on chromosome 19 codes for invariant part • Single gene on chr 9 codes for: • A GalNActransferase in the A case • A Gal transferase in the B case • A nonfunctional frameshift in O case • AB people have 1 A on one copy of chr 9 and 1 B on the other copy of chr 9 Glycolysis

10. Incompatibilities • O blood can be donated to anyone • AB people can receive any blood • People with O blood will raise IgM antibodies against A or B blood • People with B blood will raise IgM antibodies against A blood • People with A blood will raise IgM antibodies against B blood Glycolysis

11. Distributions of blood types • Note that O is the most common in general, but its prevalence varies worldwide • People with O blood are slightly more prone to infection by Vibrio cholerae Glycolysis

12. N-linked oligosaccharides • Generally linked to Asn • Types: • High-mannose • Complex (Sialic acid, …) • Hybrid(Gal, GalNAc, Man) Diagram courtesy Oregon State U. Glycolysis

13. iClicker question #1 1. Peptidoglycans in bacterial cell walls often contain one or more D-amino acids. Why? • (a) errors in transcription • (b) to make the cell walls more resistant to proteases • (c) Errors in translation • (d) uncontrolled activity of racemases • (e) none of the above Glycolysis

14. iClicker question #2 2. An N-linked oligosaccharide in a glycoprotein is • (a) never branched • (b) only attached to glutamines • (c) almost always attached to an asn • (d) highly resistant to hydrolase activity Glycolysis

15. Glycolysis (CF&M §17.1-4) • Now we’re ready for the specifics of metabolism • Why glycolysis first? • Well-understood (?) early on • Illustrates concepts used later • Inherently important Glycolysis

16. The big picture • Conversion of glucose to pyruvate • Catabolic, ten steps, energy-yielding • Overall reaction: glucose + 2 ADP + 2 NAD+ + 2Pi2pyruvate + 2ATP + 2NADH + 2H+ + 2H2O Glycolysis

17. Significance • Why is this important? • Energy production(ATP and NADH) • Pyruvate as precursor to various metabolites • Some steps require energy • So it isn’t all energy-yielding • The net reaction yields energy Glycolysis

18. The reactions • Wide variety of enzyme sizes • All the enzyme structures have been determined by X-ray crystallography, mostly at high resolution • Pentagons let you keep track of which reaction & enzyme we’re discussing n Glycolysis

19. The pathway through TIM 1 2 Figure Courtesy U.Texas 3 4 5 Glycolysis

20. Pathway: TIM to pyruvate 7 Bottom half of same graphic. Remember why all those 2’s are here! 6 8 10 9 Glycolysis

21. 1 Hexokinase + ATP-4 • Transfers γ-phosphoryl group of ATP to oxygen atom at C-6 of glucose, producing glucose 6-phosphate and ADP. • Coupling between ATP hydrolysis and an energy-requiring reaction is very close: phosphate is transferred directly from ATP to the recipient molecule, in this case glucose. + ADP-3 Glycolysis

22. Thermodynamics • Reaction catalyzed by hexokinase is energetically favored:Go’ = -22.3 kJ/mol • Reaction is subject to substantial allosteric control: it’s product-inhibited (see below) Arabidopsis hexokinaseEC 2.7.1.152kDa monomerPDB 4QS8, 1.8Å Glycolysis

23. 2 Glucose 6-P isomerase(phosphohexoseisomerase) • Interconverts glucose 6-P & fructose. • Proceeds through (1,2) ene-diol intermediate in open-chain forms Glycolysis

24. What the enzyme does • With enzyme present the energy barriers around this ene-diol are lowered enough to speed the interconversion. Francisella G6P isomerase EC 5.3.1.9125kDa dimer;monomer shownPDB 3LJK, 1.48Å Glycolysis

25. 3 Fructose 6-P + ATP → Fructose 1,6-bisP + ADP Phosphofructokinase-1 • catalyzes ATP-dependent phosphorylation at the 1 position of fructose 6-phosphate. • example of a kinase that acts on an already-phosphorylated form,creating a bisphosphorylated compound. Glycolysis

26. PFK-1 allostery • ADP acts as an allosteric activator on this enzyme as well as being a product of the reaction. • We’ll examine the control of this reaction some more later in this lecture Lactobacillus PFK-1E.C. 2.7.1.11 136K tetramer; monomer shownPDB 1ZXX, 1.85Å Glycolysis

27. 4 Fructose-1,6-bisP aldolase • Catalyzes actual C-C bond cleavage: F1-6 bisP DHAP + glyceraldehyde-3-P • Large, important enzyme • Some bacterial, yeast forms need divalent cation as a cofactor; eukaryotic aldolases do not. • Non-metal forms proceed through an imine (Schiff-base) intermediate. M.tuberculosis aldolase151 kDa tetramermonomer shownEC 4.1.2.13PDB 3ELF, 1.31Å Glycolysis

28. F-1-P Secondary activity • Enzyme is active on fructose 1-phosphate as well as its "standard" substrate, fructose 1,6-bisphosphate • in this context it forms part of catabolic pathway by which fructose itself can be used as an energy and carbon source. F-1,6 bisP Glycolysis

29. 5 Triosephosphate isomerase • Interconverts two 3-Cphosphosugars:glyceraldehyde 3-P  dihydroxyacetone P • possibly the most efficient enzyme known, in terms of the rate acceleration afforded by the enzyme relative to the uncatalyzed reaction. Glycolysis

30. TIM and TIM barrels • TIM in eukaryotes is a tetrameric enzyme with a characteristic structure in which alpha helical stretches alternate with beta strands such that the beta strands curve around to form a barrel-like structure with the helices outside. Leishmania TIM56 kDa dimermonomer shown PDB 2VXN 0.82Å; EC 5.3.1.1 Glycolysis

31. Ubiquity of TIM barrels • This structural motif appears in many other enzymes, and has become known as a "TIM barrel.” Human ribulose 5-P epimeraseEC 5.1.3.153kDa monomerPDB 3OVP, 1.7Å Glycolysis

32. 6 Glyceraldehyde-3P to1,3 bisphosphoglycerate • Reaction isD-glyceraldehyde-3-P + NAD+ + Pi1,3-bisphosphoglycerate + NADH + H+ • So we’re oxidizing glyceraldehyde and phosphorylating it without using ATP Glycolysis

33. GAPDH / G3PDH • Enzyme is glyceraldehyde 3-phosphate dehydrogenase (GAPDH or G3PDH) • Remember this buys us 2 NADH per glucose because we have 2 D-glyceraldehydes per glucose Glycolysis

34. GAPDH mechanism • Covalent catalysis (via cysteine thiohemiacetal) andacid-base catalysis • Protein-SH + Glyc3P  thiohemiacetal • NAD+ + thiohemiacetal NADH + thioester • Thioester + Pi 1,3BPG + Protein-SH Glycolysis

35. GAPDH structure • Tetrameric protein • Typical Rossmann-fold NAD-binding structure • Substantial quantity of both helix and sheet • Active-site cysteine in one of the sheet regions Thermus GAPDHE.C. 1.2.1.12145 kDa tetramerPDB 2G82, 1.65Å Glycolysis

36. 7 Phosphoglycerate kinase + ADP • catalyzes dephosphorylationof 1,3-bisphosphoglycerateto form 3-phosphoglycerate • named for reaction runningin opposite direction relativeto the one shown in chart. • During glycolysis it produces ATP rather than consuming it +ATP Glycolysis

37. PGK Structure • Has a hinge motion about a point near the center of the molecule; the open and closed forms of the enzyme involve movements as large as 17Å in the residues farthest from the hinge point. • Enzyme is primarily alpha-helical in conformation. T.thermophilus PGK EC 2.7.2.3PDB 1V6S, 1.5Å 84 kDa dimer;monomer shown Glycolysis

38. 8 Phosphoglycerate mutase • interconverts 3-phosphoglycerate and 2-phosphoglycerate • Mechanism involves formation of 2,3-bisphosphoglycerate via transient phosphorylation of a histidine Glycolysis

39. Problem with PG Mutase • 2,3BPG can diffuse from phosphoglyceratemutase, leaving the enzyme trapped in an unusable state. • Cells make excess 2,3BPG (using the enzyme bisphosphoglyceratemutase) in order to drive 2,3BPG back to phosphoglyceratemutase, so the reaction can go to completion. E.coli PGMEC 5.4.2.157 kDa dimermonomer shownPDB 1E58, 1.25Å Glycolysis

40. 9 Enolase • dehydrates 2-phosphoglycerate to phosphoenolpyruvate • This reaction plays a role in gluconeogenesis as well as glycolysis. Glycolysis

41. Enolase details • Mg2+ ions are required for activity in some forms of the enzyme. • Vertebrate genes code for two slightly different forms of the monomer of enolase, alpha and beta (and gamma…) Human -enolase EC 4.2.1.11 96 kDa dimerPDB 2AKZ, 1.36Å Glycolysis

42. Human enolase isozymes • Most of the enolase in fetal tissue is alpha-alpha; mature skeletal muscle contains beta-beta; some alpha-alpha remains in smooth muscle tissue. Glycolysis

43. 10 Pyruvate kinase • transfers a phosphate from phosphoenolpyruvate to ADP,producing pyruvate and ATP • The reaction is essentially irreversible Glycolysis

44. Regulation of Pyruvate kinase • Fructose 1,6-bisphosphate, the substrate for the aldolase reaction, is feed-forwardactivator of the reaction Human M2 pyruvate kinaseEC 2.7.1.40244 kDa tetramerPDB 3GR4, 1.6Å Glycolysis

45. What happens to pyruvate? • Pyruvate + HS–CoA + NAD Acetyl CoA + NADH + H+ • Pyruvate + HCO3- + ATP Oxaloacetate + ADP + Pi • Pyruvate + H+ Acetaldehyde + CO2Acetaldehyde + NADH + H+ Ethanol + NAD+ • Pyruvate + NADH + H+ Lactate + NAD+ Glycolysis

46. Glycolysis & free energy • General principle:For a reaction to move forward, G < 0. • That doesn’t necessarily mean Go < 0. • Irreversible steps 1,3,10.Other steps have G ≈ 0 => reversible Glycolysis

47. G and Go Glycolysis

48. Regulation of glycolysis • Thoroughly studied • Recently recognized that a compound isn’t really an effector if it modulates a reaction only when it’s much more concentrated than it ever gets in the cell • So we need to know concentrations! Glycolysis

49. What does the cell want? • Glycolysis should be turned on when ATP is needed • Usual arguments regarding products and downstream products Glycolysis

50. Heavily regulated steps • Hexokinase is product-inhibited • PFK1 has up & down effectors • Pyruvate kinase is: • feed-forward activated by F-1,6-bisP • Inhibited by ATP Glycolysis