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General Metabolism Principles; Nutrition

General Metabolism Principles; Nutrition. Andy Howard Introductory Biochemistry 1 December 2009. Metabolism depends strongly on cofactors.

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General Metabolism Principles; Nutrition

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  1. General Metabolism Principles; Nutrition Andy HowardIntroductory Biochemistry 1 December 2009 Biochemistry: Metabolism I

  2. Metabolism depends strongly on cofactors • We’ll attend to the reality that a lot of the versatility of enzymes depends on their incorporation of cofactors; and most vitamins are precursors of cofactors Biochemistry: Metabolism I

  3. Post-translational modification Phosphorylation Other reversible PTMs How pathways evolve Oxidation-Reduction Reactions: Quantitation How we study metabolism (revisited) Nutrition Macronutrients Micronutrients Specific cofactors and vitamins reconsidered Fat soluble vitamins Ascorbate What we’ll discuss Biochemistry: Metabolism I

  4. 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 • Example:glycogen phosphorylase is activated by phosphorylation; it’s a catabolic enzyme Biochemistry: Metabolism I

  5. 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 Biochemistry: Metabolism I

  6. Other PTMs • Are there other reversible post-translational modifications that regulate enzyme activity? Yes: • Adenylation of Y • ADP-ribosylation of R • Uridylylation of Y • Oxidation of cysteine pairs to cystine • Cis-trans isomerization of prolines Biochemistry: Metabolism I

  7. Metabolism and evolution • Metabolic pathways have evolved over hundreds of millions of years to work efficiently and with appropriate controls Biochemistry: Metabolism I

  8. 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 Biochemistry: Metabolism I

  9. 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 Biochemistry: Metabolism I

  10. Evolving a branch E1 E2 E3 • Original pathway: D A  B  C X • Fully evolved pathway: D A  B  C X E3a E3b Biochemistry: Metabolism I

  11. 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 Biochemistry: Metabolism I

  12. 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) Biochemistry: Metabolism I

  13. 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 Biochemistry: Metabolism I

  14. 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) Biochemistry: Metabolism I

  15. 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 Biochemistry: Metabolism I

  16. 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 Biochemistry: Metabolism I

  17. 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 Biochemistry: Metabolism I

  18. 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 Biochemistry: Metabolism I

  19. 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 Biochemistry: Metabolism I

  20. 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 Biochemistry: Metabolism I

  21. 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! Biochemistry: Metabolism I

  22. 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 I

  23. 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 Biochemistry: Metabolism I

  24. 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. Biochemistry: Metabolism I

  25. 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 Biochemistry: Metabolism I

  26. 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 Biochemistry: Metabolism I

  27. 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 I

  28. Macronutrients • Proteins • Carbohydrates • Lipids • Fiber Biochemistry: Metabolism I

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

  30. 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 I

  31. AA moles essen- ATP tial? Asp 21 no Asn 22-24 no Lys 50-51 yes Met 44 yes Thr 31 yes Ala 20 no Val 39 yes Leu 47 yes Ile 55 yes Glu 30 no Gln 31 no AA moles essen- ATP tial? Arg 44 no Pro 39 no Ser 18 no Gly 12 no Cys 19 no Phe 65 yes Tyr 62 no* Trp 78 yes His 42 yes The human list Biochemistry: Metabolism I

  32. 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 I

  33. 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 I

  34. 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 lipid vitamins first,then water-soluble vitamins Biochemistry: Metabolism I

  35. 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 I

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

  37. 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 arachidonate aren’t considered vitamins Biochemistry: Metabolism I

  38. Lipid vitamins • Contain rings & long aliphatic sidechains • At least one polar group in each • Absorbed in intestine, carried via bile salts • Hard to study • Most are formally built from isoprene units, as are steroids Biochemistry: Metabolism I

  39. Vitamin A (retinol) • 3 forms varying in terminal polar group • Involved in signaling and receptors • b-carotene is nonpolar dimer Biochemistry: Metabolism I

  40. Vitamin A deficiency • Produces night blindness because the retina and cornea dry out • Most common cause: nursing infants whose mothers have vitamin A deficiency in their diet Biochemistry: Metabolism I

  41. Vitamin D • Several related forms • Hormones involved in Ca2+ regulation Figure courtesyCyberlipid (cholecalciferol) Biochemistry: Metabolism I

  42. Vitamin D deficiency • Rickets in children:Bone disease, restlessness, slow growth • One form of vitamin D is actually synthesizable from cholesterol given adequate sunlight; • Therefore rickets is most common in densely settled urban environments Biochemistry: Metabolism I

  43. Vitamin E (a-tocopherol) • Phenol can undergo 1e- oxidation to moderately stable free radical • Antioxidant activity prevents damage to fatty acids in membranes phenol Fig. CourtesyUIC pharmacy program Biochemistry: Metabolism I

  44. Vitamin K (phylloquinone) • Involved in synthesis of proteins involved in blood coagulation • Reduced form involved as reducing agent in carboxylation reaction on glu sidechains Figure courtesyCyberlipid Biochemistry: Metabolism I

  45. Vitamin overdoses? • It’s difficult to overdose on water-soluble vitamins: excess is simply excreted • Fat-soluble vitamins are stored in adipose tissue and can accumulate to high concentrations • May be toxic even dietarily • Therefore: don’t eat polar bear liver Biochemistry: Metabolism I

  46. Ascorbate • The only common water-soluble vitamin that is not a coenzyme or coenzyme precursor • Vitamin in primates, some rodents • Synthesizable in most other vertebrates • Involved in collagen • Reduced form acts as reducing agent during hydroxylation of collagen • Deficiency gives rise to inadequate collagen - scurvy Biochemistry: Metabolism I

  47. PTM role of ascorbate • Proline + O2 + -ketoglutarate + ascorbate  4-hydroxyproline + succinate + CO2 + dehydroascorbate • This is a post-translational modification that occurs to prolines within collagen • The hydroxylated prolines help stabilize the collagen triple helix • Hydroxylysine found in collagen too Biochemistry: Metabolism I

  48. Dietary deficiency of ascorbate • Primary sources of ascorbate are fruits, particularly citrus, and green vegetables • Ascorbate deficiency’s first symptom involves collagen degradation, leading to scurvy Image courtesy U.Cincinnati Medical School Biochemistry: Metabolism I

  49. Scurvy in history • Shortage of green vegetables in sailors’ diets meant scurvy was rampant on shipboard until the 18th century • Success of English navy over French 1760-1800 was partly due to the introduction of limes in English sailors’ diets 50 years before the French caught on Biochemistry: Metabolism I

  50. Megadoses of ascorbate • Linus Pauling (2-time Nobel laureate) became convinced late in his life that very high doses of ascorbate (> 1 g /day) were beneficial as a preventative • His assertions were met with skepticism from the established medical community • I would say the jury is still out! Linus Pauling Image courtesy Oregon State U. Biochemistry: Metabolism I

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