Metabolism II: Glycolysis

1. Metabolism II:Glycolysis Andy HowardBiochemistry Lectures, Fall 201017 October 2010 Metabolism; Glycolysis

2. Metabolism Evolution of pathways Compartmentation Thermodynamics ATP High-energy compounds Redox and Energy Glycolysis Overview Steps through TIM Steps to pyruvate What we’ll discuss Metabolism; Glycolysis

3. Logistics • Midterm answer key will be posted as soon as I’m sure that everyone has taken the exam • Grading should be finished by Saturday • Next Monday’s class (10/25) will be held on Thursday morning; details to follow Metabolism; Glycolysis

4. Evolution of pathways:how have new pathways evolved? • Add a step to an existing pathway • Evolve a branch on an existing pathway • Backward evolution • Duplication of existing pathway to create related reactions • Reversing an entire pathway Metabolism; Glycolysis

5. Adding a step Original pathway E1 E2 E3 E4 E5 A  B  C  D  E P • When the organism makes lots of E, there’s good reason to evolve an enzyme E5 to make P from E. • This is how asn and gln pathways (from asp & glu) work Metabolism; Glycolysis

6. Evolving a branch E3 E1 E2 • Original pathway: D A  B  C X • Fully evolved pathway: D A  B  C X E3a E3b Metabolism; Glycolysis

7. Backward evolution • Original system has lots of E  P • E gets depleted over time; • need to make it from D, • so we evolve enzyme E4 to do that. • Then D gets depleted; • need to make it from C, • so we evolve E3 to do that • And so on Metabolism; Glycolysis

8. Duplicated pathways • Homologous enzymes catalyze related reactions;this is how trp and his biosynthesis enzymes seem to have evolved • Variant: recruit some enzymes from another pathway without duplicating the whole thing (example: ubiquitination) Metabolism; Glycolysis

9. Reversing a pathway • We’d like to think that lots of pathways are fully reversible • Usually at least one step in any pathway is irreversible (Go’ < -15 kJ mol-1) • Say CD is irreversible so E3 only works in the forward direction • Then D + ATP C + ADP + Pi allows us to reverse that one step with help • The other steps can be in common • This is how glycolysis evolved from gluconeogenesis Metabolism; Glycolysis

10. Enzyme organization • Enzymatic reactions are organized into pathways, where reactions proceed in an ordered sequence leading from the first reactant to the final product • Often, especially in eukaryotes, the relevant enzymes are spatially organized into groupings that allow one enzyme to emit its product in a position where it can be immediately picked up as a substrate by the next enzyme (G&G fig. 17.5) • These grouped enzymes are often membrane-bound to provide physical stability • Metabolons are stable multienzyme complexes that work this way Metabolism; Glycolysis

11. Compartmentation I:Localized pathways • Some are in membranes, some free in cytosol or in aqueous organelles • Several catabolic pathways in eukaryotes are localized in mitochondria • Corresponding anabolic pathways are in cytosol • Reduces likelihood of futile cycling • Multienzyme complexes, especially in eukaryotes • Provide entropic advantage • Often membrane-associated Metabolism; Glycolysis

12. Compartmentation II:Tissue Specialization • Obvious in multicellular eukaryotes • Individual cells may perform a limited number of metabolic roles • Some fully mature cells are anuclear • Requires careful cell-cell communication • Even in cyanobacteria Metabolism; Glycolysis

13. Thermodynamics • We did this carefully earlier:this is just a reminder • Remember that G is not Go’:G = Go’ + RT ln[products]/[reactants] • At equilibrium G = 0,so we can use that equation to find Go’ Metabolism; Glycolysis

14. Practical biochemical thermodynamics • Most reactions are considered either irreversible or fully reversible • Irreversible means Go’ < -20 kJ/mol; • Even with substrate and product concentrations considered, the reaction proceeds in only one direction • Reversible: -15 < Go’ < 15 butG very close to zero Metabolism; Glycolysis

15. ATP • Both the anhydride bonds are considered high-energy bonds; the phosphoester bond is not. • Remember the story about pyrophosphate!:PPi + H2O  2Pi, Go’ = -29 kJ mol-1 • Rapidity of this hydrolysis drives reactions involving pyrophosphate to right Metabolism; Glycolysis

16. ATP chemistry • Why is ATP a high-energy compound? • Negative charges repel!(somewhat mitigated by Mg2+) • ADP and Pi or AMP and PPi are better solvated than ATP • More delocalization in products • Therefore ATP (and CTP, GTP, …)are high-energy compounds Metabolism; Glycolysis

17. Interconversions among nucleotide phosphates • Kinases or phospotransferases involved • Nucleoside monophosphate kinases:ATP + XMP  ADP + XDPspecific to each X (G or dG, C or dC, …) • Nucleoside diphosphate kinase:ATP + XDP  ADP + XTPthis is a single enzyme (why?) Metabolism; Glycolysis

18. AnP interconversions • Adenylate kinase (special case of NMK…) • AMP + ATP  2ADP • [ATP] >> [ADP] and [ATP] >> [AMP], so small changes in [ATP] can drive big changes in others: [ATP],mM [ADP],mM [AMP],mM G1,kJmol-1 4.8 0.2 0.004 -40 4.5 0.5 0.02 -37 3.9 1.0 0.11 -35 3.2 1.5 0.31 -34 Metabolism; Glycolysis

19. How coupling works • We tend to hand-wave about this • Enzymes provide location for intermediates • X + ATP  X—P + ADPX—P + Y + H2O  X-Y + Pi + H+ • The X here can be an enzyme side-chain or a substrate • In the former case some other event must come along to recreate X;otherwise, it isn’t an enzyme! Metabolism; Glycolysis

20. Glutamine synthetase • Cf. section 25.2 - 25.3: • glu + ATP -glutamylphosphate + ADP • -glutamylphosphate + NH3 -> gln + Pi • Why do we need ATP at all for this? • Go’ = 14 kJ mol-1 for glu + NH3 -> gln + H2O • we could overcome that with concentrations • But we can’t: we need [glu] ~ [gln] • So we need the energy charge Metabolism; Glycolysis

21. CompoundGo’hyd, kJ mol-1 PEP -62 1,3-bisPglycerate -49 ATP->AMP+PPi -45 Phosphocreatine -43 P-arginine -32 CompoundGo’hyd Acetyl CoA* -32 ATP -32 Pyrophosphate -29 Glucose 1-P -21 Glucose 6-P -14 Glycerol 3-P -9* not a phosphate cmpd! Go’hyd for metabolites Transfers are more common than hydrolysis; these help with bookkeeping Metabolism; Glycolysis

22. Making ATP by transfer • We’ve just seen that some compounds have higher-energy phosphates than ATP • Remember the 35-cent analogy • So a phosphoryl-group transfer into ATP can be energetically favorable, e.g. Phosphocreatine + ADP  creatine + ATP Metabolism; Glycolysis

23. Relative energies of phosphate compounds • Both phosphoenolpyruvate(~ 60 kJ mol-1) and phosphocreatine(~ 45 kJ mol-1) are higher-energy than ATP (*what does that mean?) • ATP is therefore intermediate between these high-energy phosphates and the low-energy phosphates like glucose-6-phosphate and glucose-1-phosphate Metabolism; Glycolysis

24. ATP and the energy cycle • Catabolism usually gives rise to energy that is captured in high-energy phosphate bonds in ATP • This ATP is used to provide energy for otherwise endergonic reactions and to phosphorylate things that need to be phosphorylated Metabolism; Glycolysis

25. Nucleotidyl-group transfer • It’s most convenient to think of this as a transfer of the entire nucleotide group to form an acyl-adenylate intermediate • This can then fall apart, releasing AMP and allowing a high-energy bond to form • Example: acetyl CoA (see pp.561-562) Metabolism; Glycolysis

26. Thioesters: another class of high-energy compounds • Thioesters have similar reactivity as oxygen-acid anhydrides • Thioesters less stable than oxygen esters because the unshared electrons in sulfur are not as delocalized in a thioester as the unshared electrons in an oxygen ester Metabolism; Glycolysis

27. Oxidation-reduction reactions and Energy • Oxidation-reduction reactions involve transfer of electrons, often along with other things • Generally compounds with many C-H bonds are high in energy because the carbons can be oxidized (can lose electrons) Metabolism; Glycolysis

28. Reduction potential • Reduction potential is a measure of thermodynamic activity in the context of movement of electrons • Described in terms of half-reactions • Each half-reaction has an electrical potential, measured in volts, associated with it because we can (in principle) measure it in an electrochemical cell Metabolism; Glycolysis

29. So what is voltage, anyway? • Electrical potential is available energy per unit charge: • 1 volt = 1 Joule per coulomb • 1 coulomb = 6.24*1018 electrons • Therefore energy is equal to the potential multiplied by the number of electrons Metabolism; Glycolysis

30. Electrical potential and energy • This can be expressed thus:Go’ = -nFEo’ • n is the number of electrons transferred • F = fancy way of writing # of Coulombs (which is how we measure charge) in a mole (which is how we calibrate our energies) = 96.48 kJ V-1mol-1 Metabolism; Glycolysis

31. Oh yeah? • Yes. • 1 mole of electrons = 6.022 * 1023 e- • 1 coulomb = 6.24*1018 e- • 1 mole = 9.648*104 Coulomb • 1 V = 1 J / Coulomb=10-3 kJ / Coulomb • Therefore the energy per mole associated with one volt is10-3 kJ / C * 9.648*104 C = 96.48 kJ Metabolism; Glycolysis

32. What can we do with that? • The relevant voltage is the difference in standard reduction potential between two half-reactions • Eo’ = Eo’acceptor - Eo’donor • Combined with free energy calc, we seeEo’ = (RT/nF ) lnKeq andE = Eo’ - (RT/nF ) ln [products]/[reactants] • This is the Nernst equation Metabolism; Glycolysis

33. Free energy from electron transfer • We can examine tables of electrochemical half-reactions to get an idea of the yield or requirement for energy in redox reactions • Example (see section 10.9B):NADH + (1/2)O2 + H+ -> NAD+ + H2O; • We can break that up into half-reactions to determine the energies Metabolism; Glycolysis

34. Half-reactions and energy • NAD+ + 2H+ + 2e- NADH + H+,Eo’ = -0.32V • (1/2)O2 + 2H+ + 2e- H2O, Eo’ = 0.82V • Reverse the first reaction and add:NADH + (1/2)O2 + H+  NAD+ + H2O,Eo’ = 0.82+0.32V = 1.14 V. • Go’ = -nFEo’ = -2*(96.48 kJ V-1mol-1)(1.14V) = -220 kJ mol-1; that’s a lot! Metabolism; Glycolysis

35. NAD: electron collector • Net reactions involve transfer of hydride (H:-) ions • Enzymes called dehydrogenases (a type of oxidoreductase) involved • Collected NADH can then be reoxidized in oxidative phosphorylation to drive ATP synthesis in the mitochondrion Metabolism; Glycolysis

36. NADPH • Provides reducing power for anabolic reactions • Often converting highly oxidized sugar precursors into less reduced molecules Metabolism; Glycolysis

37. NAD+ 340 nm How to detect NAD reactions Absorbance NADH Wavelength • NAD+ and NADH(and NADP+ and NADPH)have extended aromatic systems • But the nicotinamide ring absorbs strongly at 340 only in the reduced(NADH, NADPH) forms • Spectrum is almost pH-independent, too! • So we can monitor NAD and NADP-dependent reactions by appearance or disappearance of absorption at 340 nm Metabolism; Glycolysis

38. How much ATP can we get out of oxidizing NADH? • In principle -220 kJ mol-1 should be enough to drive production of at least five ATP molecules • (220/32) = 6.9; even if we figure it will cost more like 40 kJ mol-1 per ATP, then that’s (220/40) = 5.5. • But in fact we only get about 3.5. Metabolism; Glycolysis

39. Why? • Short answer: discrete inefficiency • 4 oxidation steps in the electron transport chain beginning with NADH • 3 of 4 of those steps facilitate transfer of protons against a pH and charge gradient • When those protons move back across with their charge and concentration gradients, we earn ATP back • … but only about net 3.5 ATP per NADH Metabolism; Glycolysis

40. Pathway methods • Introducing inhibitors • Site-directed mutagenesis • Radioisotope tracing • Non-radioactive isotope tracing • NMR Metabolism; Glycolysis

41. Classical metabolism studies • Add substrate to a prep and look for intermediates and end products • If substrate is radiolabeled (3H, 14C) it’s easier, but even nonradioactive isotopes can be used for mass spectrometry and NMR • NMR on protons, 13C, 15N, 31P • Reproduce reactions using isolated substrates and enzymes Metabolism; Glycolysis

42. Next level of sophistication… • Look at metabolite concentrations in intact cell or organism under relevant physiological conditions • Note that Km is often ~ [S].If that isn’t true, maybe you’re looking at the non-physiological substrate! • Think about what’s really present in the cell. Metabolism; Glycolysis

43. Mutations in single genes • If we observe or create a mutation in a single gene of an organism, we can find out what the effects on viability and metabolism are • In humans we can observe genetic diseases and tease out the defective gene and its protein or tRNA product • Sometimes there are compensating enzyme systems that take over when one enzyme is dead or operating incorrectly Metabolism; Glycolysis

44. Deliberate manipulations • Bacteria and yeast: • Irradiation or exposure to chemical mutagens • Site-directed mutagenesis • Higher organisms:We can delete or nullify some genes;thus knockout mice • Introduce inhibitors to pathways and see what accumulates and what fails to be synthesized Metabolism; Glycolysis

45. Glycolysis • Now we’re ready for the specifics of metabolism • Why glycolysis first? • Well-understood (?) early on • Illustrates concepts used later • Inherently important Metabolism; Glycolysis

46. 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 Metabolism; Glycolysis

47. 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 Metabolism; Glycolysis

48. The reactions • See fig. 11.2 and the table in the HTML notes • Wide variety of enzyme sizes • All then enzyme structures have been determined by X-ray crystallography, mostly at high resolution Metabolism; Glycolysis

49. The pathway through TIM Figure Courtesy U.Texas Metabolism; Glycolysis

50. Pathway: TIM to pyruvate Bottom half of same graphic Metabolism; Glycolysis