General Metabolism

1. General Metabolism Andy HowardIntroductory Biochemistry 3 December 2014 Biochemistry: Metabolism

2. Metabolism:the core of biochemistry • All of biology 402 will concern itself with the specific pathways of metabolism; our purpose here is to arm you with the necessary weaponry • We’ll then cover nutritional biochemistry, which fits naturally into these topics because many vitamins are precursors of coenzymes. Biochemistry: Metabolism

3. Metabolism Definitions Pathways Control Phosphorylation Other PTMs Evolution Nutritional biochemistry Macronutrients Vitamins Water-soluble Lipidic Vitamins as coenzyme precursors What we’ll discuss Biochemistry: Metabolism

4. Metabolism • Almost ready to start the specifics(chapter 18) • 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! Biochemistry: Metabolism

5. Intermediary Metabolism • Metabolism involving small 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? Biochemistry: Metabolism

6. How similar are pathways in various organisms? • Enormous degree of similarity in the general metabolic approaches all the way from E.coli to elephants • Glycolysis arose prior to oxygenation of the atmosphere • This is considered strong evidence that all living organisms are derived from a common ancestor Biochemistry: Metabolism

7. Anabolism and catabolism • Anabolism: synthesis of complex molecules from simpler ones • Generally energy-requiring • Involved in making small molecules and macromolecules • Catabolism: degradation of large molecules into simpler ones • Generally energy-yielding • All the sources had to come from somewhere Biochemistry: Metabolism

8. 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 Biochemistry: Metabolism

9. Metabolism and energy Biochemistry: Metabolism

10. Metabolic classifications • Carbon sources • Autotrophs vs. heterotrophs • Atmospheric CO2 as a C source vs. otherwise-derived C sources • Energy sources • Phototrophs vs. chemotrophs • (Sun)light as source of energy vs. reduced organic compounds as a source of energy Biochemistry: Metabolism

11. Fourway divisions (table 17.2) Biochemistry: Metabolism

12. Another distinction: the organism’s interaction with oxygen • Aerobes: use O2 as the ultimate electron acceptor in oxidation-reduction reactions • Anaerobes: don’t depend on O2 • Obligate: poisoned by O2 • Facultative: can switch hit Biochemistry: Metabolism

13. Flow of energy • Sun is ultimate source of energy • Photoautotrophs drive synthesis of [reduced] organic compounds from atmospheric CO2 and water • Chemoheterotrophs use those compounds as energy sources & carbon; CO2 returned to atmosphere Biochemistry: Metabolism

14. How to anabolism & catabolism interact? • Sometimes anabolism & catabolism occur simultaneously. • How do cells avoid futile cycling? • Just-in-time metabolism • Compartmentalization: • Anabolism often cytosolic • Catabolism often mitochondrial Biochemistry: Metabolism

15. 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 • Branched • Circular Biochemistry: Metabolism

16. Catabolism stages • Stage 1: big nutrient macromolecules hydrolyzed into their building blocks • Stage 2: Building blocks degraded into limited set of simpler intermediates, notably acetyl CoA • Stage 3: Simple intermediates are fed to TCA cycle and oxidative phosphorylation Biochemistry: Metabolism

17. Anabolism stages • Short list ofsimple precursors • These are elaboratedin characteristic ways to build monomerse.g.: transamination of -ketoacids to make -amino acids • Those are then polymerized to form proteins, polysaccharides, polynucleotides, etc. transamination Biochemistry: Metabolism

18. Some intermediates play two roles • Some metabolites play roles in both kinds of pathways • We describe them as amphibolic • Just recall that:catabolism is many down to few, anabolism is few up to many Biochemistry: Metabolism

19. Anapleurotic reactions • Certain metabolites that are integral to particular pathways also get used in other places • The result is that the concentration of those metabolites may get depleted by the competing pathways • The cell may need to replenish those metabolites in order to keep the original pathway humming • We describe reactions that replenish metabolites as anapleurotic Biochemistry: Metabolism

20. Differences between catabolic and anabolic pathways • Often they share many reactions, notably the ones that are nearly isoergic (Go ~ 0) • Reactions with Go < -20 kJ mol-1 are not reversible as is • Those must be replaced by (de)coupled reactions so that the oppositely-signed reactions aren’t unfeasible Biochemistry: Metabolism

21. Other differences involve regulation • Generally control mechanisms influence catalysis in both directions • Therefore a controlling influence(e.g. an allosteric effector)will up- or down-regulate both directions • If that’s not what the cell needs, it will need asymmetric pathways or pathways involving different enzymes in the two directions Biochemistry: Metabolism

22. ATP’s role • We’ve discussed its significance as an energy currency • It’s one of two energy-rich products of the conversion of light energy into chemical energy in phototrophs • ATP then provides drivers for almost everything else other than redox Biochemistry: Metabolism

23. NAD’s role • NAD acts as asan electronacceptor via nettransfer of hydride ions,H:-, in catabolic reactions • Reduced substrates get oxidized in the process, and their reducing power ends up in NADH • Energy implied by that is used to make ATP (2.5 ATP/NAD) in oxidative phosphorylation Image courtesy Michigan Tech Biological Sciences Biochemistry: Metabolism

24. NADPH’s role • Involved in anabolic redox reactions • Reducing power in NADPH  NADP used to reduce some organic molecule • Involves hydride transfers again • NADPH regenerated in phototrophs via light-dependent reactions that pull electrons from water Biochemistry: Metabolism

25. 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 Biochemistry: Metabolism

26. How do we study pathways? • Inhibitor studies • Mutagenesis • Isotopic tracers (radio- or not) • NMR • Disruption of cells to examine which reactions take place in which organelle Biochemistry: Metabolism

27. Why multistep pathways? • Narrow 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 Biochemistry: Metabolism

28. iClicker quiz question 1 A reaction A+B  C+D proceeds from left to right in the cytosol and from right to left in the mitochondrion. As written, it is probably • (a) a catabolic reaction • (b) an anabolic reaction • (c) an amphibolic reaction • (d) we don’t have enough information to answer. Biochemistry: Metabolism

29. iClicker quiz question 2 • An asymmetry between stage 1 of catabolism (C1) and the final stage of anabolism (A3) is • (a) A3 always requires light energy;C1 doesn’t • (b) A3 never produces nucleotides;C1 can involve nucleotide breakdown • (c) A3 adds one building block at a time to the end of the growing polymer;C1 can involve hydrolysis in the middle of the polymer • (d) There are no asymmetries between A3 and C1 Biochemistry: Metabolism

30. iClicker quiz question 3 • Could dAMP, derived from degradation of DNA, serve as a building block to make NADP? • (a) Yes. • (b) Probably not: the energetics wouldn’t allow it. • (c) Probably not: the missing 2’–OH would make it difficult to build NADP • (d) No: dAMP is never present in the cell Biochemistry: Metabolism

31. Nutrition • Lots of nonsense,some sense on this subject • Skepticism among MDs as to its relevance • Fair view is that nutrition matters in many conditions, but it’s not the only determinant of health Biochemistry: Metabolism

32. Macronutrients • Proteins • Carbohydrates • Lipids • Fiber Biochemistry: Metabolism

33. Protein as food • Source of essential amino acids • Source of non-essential amino acids • Fuel (often via interconversion to -ketoacids and incorporation into TCA) • All of the essential amino acids must be supplied in adequate quantities Biochemistry: Metabolism

34. Which amino acids are essential? • At one level, that’s an easy question to answer: they’re the ones for which we lack a biosynthetic pathway: KMTVLIFWH • That shifts the question to:why have some of those pathways survived and not all? • Answer: pathways that are complex or require more than ~30 ATP / aa are absent (except R,Y) Biochemistry: Metabolism

35. Essential & non-essential aa’s Non-essential A.A. name # ATP’s • Glycine 12 • Serine 18 • Cysteine 19 • Alanine 20 • Aspartate 21 • Asparagine 22-24 • Glutamate 30 • Glutamine 31 • Proline 39 • Arginine 44 * • Tyrosine 62 ** Essential A.A. Name #ATPs • Threonine 31 • Valine 39 • Histidine 42 • Methionine 44 • Leucine 47 • Lysine 50-51 • Isoleucine 55 • Phenylalanine 65 • Tryptophan 78 * Essential in some organisms** Derived from phenylalanine Biochemistry: Metabolism

36. Carbohydrates as food • Generally recommended to be more than half of caloric intake • Complex carbohydrates are hydrolyzed to glucose-1-P and stored as glycogen or interconverted into other metabolites Biochemistry: Metabolism

37. Lipids as food • You’ll see in 402 that the energy content of a lipid is ~ 2x that of carbohydrates simply because they’re more reduced • They’re also more efficient food storage entities than carbs because they don’t require as much water around them • Certain fatty acids are not synthesizable; by convention we don’t call those vitamins Biochemistry: Metabolism

38. Vitamins • Vitamins are necessary micronutrients • A molecule that is a vitamin in one organism isn’t necessarily a vitamin in another • E.coli can make all necessary metabolites given sources of water, nitrogen, and carbon • Most eukaryotic chemoautotrophs find it more efficient to rely on diet to make complex metabolites • We’ll discuss water-soluble vitamins first, then lipid vitamins Biochemistry: Metabolism

39. Why wouldn’t organisms make everything? • Complex metabolites require energy for synthesis • Control of their synthesis is also metabolically expensive • Cheaper in the long run to derive these nutrients from diet Biochemistry: Metabolism

40. Vitamins: broad classifications • Water-soluble vitamins • Coenzymes or coenzyme precursors • Non-coenzymic metabolites • Fat-soluble vitamins • Antioxidants • Other lipidic vitamins Biochemistry: Metabolism

41. Are all nutrients that we can’t synthesize considered vitamins? • No: • If it’s required in large quantities,it’s not a vitamin • By convention, essential fatty acids like linoleate aren’t considered vitamins Biochemistry: Metabolism

42. Warning: ugly photos coming • I have included some web-derived photos of patients with severe vitamin deficiencies • If you’re squeamish, be prepared. Biochemistry: Metabolism

43. Coenzyme precursors • We’ve already outlined the fact that most water-soluble coenzymes are derived from vitamins—typically B vitamins • Typically the dietary form can be converted by a fairly short metabolic pathway into the coenzyme form, e.g. • niacin + glutamine nicotinamide + glutamate • nicotinamide + ADP-ribose  NAD • Some coenzyme precursors are, in fact, lipidic Biochemistry: Metabolism

44. The B vitamins • All aqueous micronutrients • Generally identified via pathologies associated with dietary deficiencies • B1: thiamin (produces TPP) • B2: riboflavin (produces FAD, FMN) • B3: niacin (produces NAD, NADP) • B5: pantothenate (produces Coenzyme A) • B6: pyridoxamine (produces PLP) • B9: folate: produces THF, THF derivatives • B12: cobalamin (produces adenosylcobalamin, methylcobalamin) Biochemistry: Metabolism

45. Deficiency of niacin (B3) • Remember: niacin is the source for NAD and NADP (redox cofactors) • Pellagra: dermatitis, diarrhea, dementia • Still found in some diets that are low in vitamin content • Humans can actually synthesize nicotinamide from tryptophan; but that’s often in short supply too Image courtesy history.nih.gov Biochemistry: Metabolism

46. Deficiency of thiamin (B1) • Remember: thiamin is precursor to TPP (used in decarboxylations) • Beriberi: primary symptoms are in nervous system &musculature • Polished rice is missing thiamine; rice hulls are rich in it Image courtesy answers.com Biochemistry: Metabolism

47. Riboflavin (Vitamin B2) • Precursor of the redox cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) • Key property: can undergo one-electron as well as two-electron redox interactions because of delocalization across the isoalloxazine aromatic ring system • Deficiencies can lead to growth retardation Biochemistry: Metabolism

48. Pantothenate (Vitamin B5) (pantoate) • Precursor of coenzyme A, critical in lipid and TCA-cycle metabolism • Made up of pantoate and beta-alanine components • Deficiency leads to dermatitis in chickens (β-alanine) Biochemistry: Metabolism

49. pyridoxine Pyridoxal (Vitamin B6) • Precursor of pyridoxal phosphate (PLP), a crucial cofactor in enzymatic reactions involving amino acids, including transaminations • Available in diet as pyridoxine, pyridoxal, pyridoxamine • Modest deficiencies lead to dermatitis in rats pyridoxal pyridoxamine Biochemistry: Metabolism

50. Biotin (Vitamin B7) • Used directly as a coenyzme in carboxylation reactions • Modest deficiency leads to dermatitis in humans • Most hydrophobic of the B vitamins Biochemistry: Metabolism