General Metabolism Overview

1. Introduction to Metabolism;Carbohydrate Chemistry I Andy HowardBiochemistry Lectures, Spring 2019Thursday 14 March 2019

2. Metabolism • We’ll look at metabolism at the 30,000-foot level • Then we’ll begin our characterization of carbohydrates General Metabolism

3. Metabolism Pathways Anabolism Catabolism Energy & Oxygen Regulation & PTM Redox & Energy Methods Carbohydrates Monomers Chirality Cyclization Sugar Derivatives What we’ll discuss General Metabolism

4. Metabolism • Almost ready to start the specifics • Define it!Metabolism is the network of chemical reactions that occur in biological systems, including the ways in which they are controlled. • So it covers most of what we do here! General Metabolism

5. Metabolic principles learned here. . . • … will be useful throughout the remainder of the course • We’ll need concepts of energy flux, feedback, feed-forward, post-translational modification, thermodynamics, kinetics, reduction potential, . . . General Metabolism

6. Intermediary Metabolism • Metabolism involvingsmall molecules • Describing it this way is a matter of perspective:Do the small molecules exist to give the proteins something to do, or do the proteins exist to get the metabolites interconverted? Chart courtesyWormbook.org General Metabolism

7. Metabolic pathways • We can understand metabolic pathways in terms of macromolecular behavior as well as small-molecule behavior. • Cofactors and vitamins are components of those pathways General Metabolism

8. Pathway • A sequence of reactions such that the product of one is the substrate for the next • Similar to an organic synthesis scheme (but with better yields!) • May be: • Unbranched (A  B  C D) • Branched • Circular General Metabolism

9. Metabolic pathways • Anabolism: buildup of complex molecules from simple ones, generally with the insertion of energy in the form of ATP hydrolysis • Catabolism: breakdown of complex molecules into simpler ones, usually with release of energy in the form of ATP production or reduction of NAD to NADH • Amphibolism: Overlap of anabolism with catabolism within one pathway General Metabolism

10. Why multistep pathways? • Limited reaction specificity of enzymes • Control of energy input and output: • Break big inputs into ATP-sized inputs • Break energy output into pieces that can be readily used elsewhere General Metabolism

11. Anabolic Pathways • Buildup of complex molecules • Specific pathways: • Gluconeogenesis (pyruvate  glucose) • Parts of TCA cycle and glyoxalate pathway • Calvin cycle • Starch and glycogen synthesis • Nucleotide and amino acid synthesis • Fatty acid and lipid synthesis General Metabolism

12. Anabolic pathways • Not all-encompassing;this is from Citizendium.org General Metabolism

13. Anabolic divergence • A few simple precursor molecules get combined and modified to form many end products • Building blocks generated from various metabolites, e.g.: • -amino acids derived by (trans)amination of -ketoacids • Fatty acids built up two carbons at a time from acetyl CoA • Carbohydrates built up from pyruvate General Metabolism

14. Catabolic pathways • Energy-yielding oxidations • Breakdown of storage molecules • Breakdown of N-containing molecules • Glucose to TCA cycle • TCA cycle • Electron transport and oxidative phosphorylation General Metabolism

15. Catabolic pathways • Again, this is not all-inclusive; • This picture from citizendium.org General Metabolism

16. Catabolism: convergence • Stage 1: break nutrients into building blocks • Stage 2: break building blocks into a very small number of end products General Metabolism

17. Note that some pathways are both anabolic and catabolic! • These pathways are amphibolic • Specific metabolites are both intermediaries in these pathways and they’re useful in other contexts • If a metabolite is depleted out of a pathway,we generally need a replenishment reaction to rebalance things • Replenishment reactions are called anapleurotic General Metabolism

18. Are build-up and breakdown identical? • No: Energetics say we can’t do that • Some enzymes (catalyzing nearly isoergic reactions) are shared • Others differ in ATP or other energy requirements • Control elements can be different too General Metabolism

19. Common metabolic themes • Maintenance of internal concentrations of ions, metabolites, enzymes • Extraction of energy from external sources • Pathways specified genetically • Organisms & cells interact with their environment • Constant degradation & synthesis of metabolites and macromolecules to produce steady state General Metabolism

20. Metabolism and energy General Metabolism

21. Energy & carbon • Whence organisms obtain their energy and their carbon defines their metabolic identity • Autotrophs • Photoautotrophs:get energy from light and C from CO2 • Chemiautotrophs (bacterial only)get energy from food and C from CO2 Methanopyrus,a chemiautotroph(Wikipedia) General Metabolism

22. Heterotrophs Theocapsa, A photoheterotrophKenyon microwiki Photoheterotrophs(bacterial only):energy from light,require organic carbon Chemoheterotrophs:energy from food,require organic carbon General Metabolism

23. Energy flow • Energy flows from the sun via photosynthesis and then flows through biological systems through the ingestion of food, generation of heat, and other thermodynamic processes Diagram courtesy Gerald Marten General Metabolism

24. The role of oxygen • Oxygen is necessary to survival in most organisms—namely, aerobic organisms—where it functions as the final electron acceptor for the electron transport chain. • It is, however, toxic because it’s reactive in ways that are often deleterious. • Many mechanisms exist for detoxifying the undesirable side-products of oxygen metabolism, particularly in aerobic organisms, where the organism can’t simply escape O2. General Metabolism

25. Regulation • Organisms respond to change • Fastest: small ions move in msec • Metabolites: 0.1-5 sec • Enzymes: minutes to days • Flow of metabolites is flux: • steady state is like a leaky bucket • Addition of new material replaces the material that leaks out through the holes Cartoon courtesycommonwealthsolar.com General Metabolism

26. Feedback and Feed-forward • Mechanisms by which the concentration of a metabolite that is involved in one reaction influences the rate of some other reaction in the same pathway General Metabolism

27. Feedback realities • Control usually exerted at first committed step (i.e., the first reaction that is unique to the pathway) • Otherwise, it occurs on irreversible steps • Controlling element is usually the last element in the path General Metabolism

28. Feed-forward • Early metabolite activates a reaction farther down the pathway • Has the potential for instabilities, just as in electrical feed-forward • Usually modulated by feedback General Metabolism

29. Activation and inactivation by post-translational modification • Most common:covalent phosphorylation of protein • usually S, T, Y, sometimes H, D, E • Kinases add phosphateProtein-OH + ATP  Protein-O-PO3 + ADP… ATP is source of energy and Pi General Metabolism

30. Phosphorylation’s effects • Phosphorylation of an enzyme can either activate it or deactivate it • Usually catabolic enzymes are activated by phosphorylation and anabolic enzymes are inactivated General Metabolism

31. Example of phosphorylation Cartoon courtesyMolecular Bioinformatics Center, Taiwan Glycogen phosphorylase is activated by phosphorylation; it’s a catabolic enzyme General Metabolism

32. Glycogen phosphorylase • Reaction: extracts 1 glucose unit from non-reducing end of glycogen & phosphorylates it:(glycogen)n + Pi(glycogen)n-1 + glucose-1-P • Activated by phosphorylationvia phosphorylase kinase • Deactivated by dephosphorylation by phosphorylase phosphatase Rabbit muscleglycogen phosphorylasePDB 2GJ4; 1.6Å192 kDa dimer; monomer shown General Metabolism

33. Amplification • Activation of a single molecule of a protein kinase can enable the activation (or inactivation) of many molecules per sec of target proteins • Thus a single activation event at the kinase level can trigger many events at the target level General Metabolism

34. Other Reversible PTMs that regulate enzyme activity • Adenylylation of tyr(gln synthetase) • ADP-ribosylation of arg (cholera toxin A1) • Uridylylation of tyr (protein-DNA crosslinks) • Oxidation of cysteine pairs to cystine(ubiquitous in oxidizing environments) Human gln synthetase221 kDa homopentamerPDB 2OJW, 2.05Å General Metabolism

35. 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) General Metabolism

36. 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 General Metabolism

37. So what is voltage, anyway? • Electrical potential is available energy per unit charge: • 1 volt = 1 Joule per coulomb • 1 coulomb = 6.24*1018 charges • So energy = potential * number of electrons • Note: we discussed this briefly when we studied membrane translocation General Metabolism

38. Electrical potential and energy • This can be expressed thus: Go’ = -nFEo’ • This is like what we did last time except we’re using electrons, for which Z=-1 but we might be moving more than one at a time. • n is the number of electrons transferred • Remember F = 96485 J/(V-coul) General Metabolism

39. 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 General Metabolism

40. 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 :NADH + (1/2)O2 + H+ NAD+ + H2O; • We can break that up into half-reactions to determine the energies General Metabolism

41. 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! General Metabolism

42. 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 General Metabolism

43. NAD hydride transfer • We said the NAD half-reaction could be written NAD++2H++2e- NADH+H+ • But if we recognize that we can think of this as a hydride (H:-) transfer, it’sNAD+ + H+ + H:- NADH + H+ • And since the protons appear on both sides, that’s NAD+ + H:- NADH General Metabolism

44. NADPH • Provides reducing power for anabolic reactions • Often converting highly oxidized sugar precursors into less oxidized molecules General Metabolism

45. 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 General Metabolism

46. 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 say it’ll cost more like 40 kJ mol-1 per ATP, then that’s (220/40) = 5.5. • But in fact we only get about 2.5. General Metabolism

47. Why do we get only 2.5? • 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 electrical potential gradient • When protons move back across with their charge and concentration gradients, we earn ATP back: only about nets 2.5 ATP per NADH General Metabolism

48. Pathway methods • Introducing inhibitors • Site-directed mutagenesis • Radioisotope tracing • Non-radioactive isotope tracing • NMR General Metabolism

49. Classical metabolism studies • Add substrate to a prep and look for intermediates and end products • If substrate is radiolabeled (3H, 14C) it’s easy, 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 General Metabolism

50. 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. General Metabolism