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Amino acid metabolism II; nucleic acid chemistry I

Amino acid metabolism II; nucleic acid chemistry I. Andy Howard Biochemistry Lectures, Fall 2010 17 November 2010. Amino acid metabolism. Amino acid synthetic pathways are complex but we can understand them if we’re patient Amino acid degradation falls into two broad categories

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Amino acid metabolism II; nucleic acid chemistry I

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  1. Amino acid metabolism II;nucleic acid chemistry I Andy HowardBiochemistry Lectures, Fall 201017 November 2010 Biochem: AAmetab 2; Nuc 1

  2. Amino acid metabolism • Amino acid synthetic pathways are complex but we can understand them if we’re patient • Amino acid degradation falls into two broad categories • Nucleic acid chemistry: N-rings Biochem: AAmetab 2; Nuc 1

  3. Amino acid synthesis Glutamate Glutamine Other simple aas Essential amino acids Amino acid catabolism Degradation products Interconversions Specifics Urea cycle Reactions Cellular localization Nucleic acid chemistry Pyrimidines: C, U, T Purines: A, G Nucleosides Nucleotides Oligo- and polynucleotides Duplex DNA Helicity What we’ll cover Biochem: AAmetab 2; Nuc 1

  4. Glutamate • Glutamate is a critical metabolite because so many of the transaminations start with it as the amine donor • It is produced in E.coli, etc. via glutamate dehydrogenase using ammonium ion as nitrogen donor:-ketoglutarate + NH4+ + NAD(P)H + H+ NAD(P)+ + H2O + glutamate 1BGV296 kDa hexamermonomer shown EC 1.4.1.2, 1.9ÅClostridium Biochem: AAmetab 2; Nuc 1

  5. Glutamine • Glutamate can be aminated with expenditure of ATP to form glutamine:glutamate + NH4+ + ATP glutamine + ADP + Pi • Note that glutamine synthetase is a ligase: the ATP is an energy-provider, not a phosphate donor Glutamine synthetase PDB 2OJW211 kDa pentamer HumanEC 6.3.1.2, 2.05Å Biochem: AAmetab 2; Nuc 1

  6. Aspartate and asparagine • Asp is simple:transamination ofoxaloacetate • Asn is straightforward too • asparagine synthetase moves the amine from gln to asp, leaving glu (another ligase) • Gln + asp + ATP AMP + PPi + glu + asn Asparagine synthetase B PDB 1CT9243 kDa EC 6.3.5.4, 2ÅtetramerE.coli Biochem: AAmetab 2; Nuc 1

  7. Simple: ala, gly, ser • Alanine by transamination from pyruvate • Glycine from serine by SHMT (q.v.) • Serine from 3-phosphoglycerate: • 3-phosphoglycerate + NAD NADH + H+ + 3-phosphohydroxypyruvate • 3-phosphohydroxypyruvate + glutamate  3-phosphoserine + -ketoglutarate • 3-phosphoserine + H2O  serine + Pi Phosphoserine phosphatasePDB 1NNL49 kDa dimerhumanEC 3.1.3.3 1.53Å Biochem: AAmetab 2; Nuc 1

  8. Serine hydroxymethyl-transferase • Serine + tetrahydrofolate H2O + glycine + 5,10-methylene-tetrahydrofolate • This can be viewed as a source of methylene units for other biosyntheses • PLP-dependent reaction SHMT PDB 2DKJ90 kDa dimerEC 2.1.2.11.15Å Thermus thermophilus Biochem: AAmetab 2; Nuc 1

  9. Glutamate semialdehyde Arginine and proline • Two routes: • Glutamate to glutamate semialdehyde • that cyclizes to 1-pyrroline 5-carboxylateand thence to proline • Glutamate semialdehyde can also be converted to ornithine and thence to arg • Alternative: glutamate acetylated to N-acetyl-glutamate-5-semialdehyde and thence to ornithine ornithine Biochem: AAmetab 2; Nuc 1

  10. Glutamate to P5C • Single enzyme can interconvert glutamate and 1-pyrroline carboxylate:1-pyrroline-5-carboxylate dehydrogenase • 3-layer  sandwich protein PDB 2BJA300 kDa hexamerdimer shownEC 1.5.1.12, 1.9ÅThermus thermophilus Biochem: AAmetab 2; Nuc 1

  11. Pyrroline-5-carboxylate to proline • Pyrroline-5-carboxylate reduced to proline • Large, NAD(P)-dependent enzyme Pyrroline-5-carboxylate reductasePDB 2IZZEC 1.5.1.2, 1.95Å354 kDa decamerpentamer shownHuman Biochem: AAmetab 2; Nuc 1

  12. Glutamate to Glu semialdehyde • Glu is -phosphorylated:glu + ATP glu-5-P +ADP (2.7.2.11) • Glu-5-P is reduced and dephosphorylated:glu-5-P + NADPH + H+ glu-5-semialdehyde + NADP+ -glutamyl phosphate reductasePDB 1O2047 kDa monomerThermatoga maritimaEC 1.2.1.41, 2Å Glu-5-P Biochem: AAmetab 2; Nuc 1

  13. Glu semialdehyde to ornithine • This is just another transamination, catalyzed by ornithine aminotransferase:glu-5-semialdehyde + glu/asp  ornithine + -keto-glutarate / oxaloacetate • Typical PLP-dependent reaction PDB 2OAT193 kDa tetramerhumanEC 2.6.1.13, 1.95Å ornithine Biochem: AAmetab 2; Nuc 1

  14. Carbamoyl phosphate citrulline Ornithine to citrulline • Ornithine condenses with carbamoyl phosphate to form citrulline with the help of ornithine transcarbamoylase PDB 1DUV110 kDa trimerE.coliEC 2.1.3.3, 1.7Å Biochem: AAmetab 2; Nuc 1

  15. Citrulline to arginosuccinate Argininosuccinate synthasePDB 2NZ2, 2.4ÅEC 6.3.4.5 200 kDa tetramermonomer shown • Citrulline condenses with aspartate using ATP hydrolysis to drive it forward to L-arginosuccinate:citrulline + aspartate + ATP  L-arginosuccinate + AMP + PPi Biochem: AAmetab 2; Nuc 1

  16. Arginosuccinate to arginine • Fumarate extracted,leaving arginine • Arginosuccinate lyase is also -crystallin, one of the moonlighting proteins: it’s a component of eye lenses PDB 1TJ7100 kDa dimerEC 4.3.2.1, 2.44ÅE.coli fumarate Biochem: AAmetab 2; Nuc 1

  17. Why all that detail? • These reactions form 75% of the urea cycle, which is an important path for amino acid and nucleic acid degradation. • So we’ll need this later. Biochem: AAmetab 2; Nuc 1

  18. Cysteine synthesis in plants and bacteria • serine + Acetyl CoA O-acetylserine + HSCoA • O-acetylserine + S2- + H+ cysteine + acetate • Ser acetyltransferase is inhibited by cysteine Serine acetyltransferasePDB 1SSQ176 kDa hexamerdimer shownHaemophilusEC 2.3.1.30, 1.85Å O-acetylserine Biochem: AAmetab 2; Nuc 1

  19. Animal pathway to cys • Ser + homocysteine (from met) fuse to form cystathionine + H2O • Cystathionine + H2O  NH4+ + cysteine + -ketobutyrate Cystathionine -lyasePDB 1N8PEC 4.4.1.1, 2.6Å 173 kDa tetrameryeast cystathionine Biochem: AAmetab 2; Nuc 1

  20. Amino acids we’ve already covered Acids and amides:glu, gln, asp, asn Simple:ala, ser, gly Other non-essential amino acids:arg, pro, cys Essential but straightforward lys, met, thr val, leu, ile Essential & Ugly phe, tyr, trp his Marching through the list of twenty amino acids Biochem: AAmetab 2; Nuc 1

  21. Lys, met, thr • asp gets phosphorylated and becomes a source for all of these: • asp + ATP-aspartyl phosphate + ADPvia aspartate kinase • -asp P + NADPH + H+ -> Pi + aspartate -semialdehyde +NADP+ • This heads to lys or to homoserine • Homoserine converts in a few steps to met or thr Aspartate kinase112 kDaEC 2.7.2.4 2.85ÅPDB 2CDQdimerArabidopsis Biochem: AAmetab 2; Nuc 1

  22. Asp -semialdehyde to homoserine • -aldehyde reduced to sec-alcohol, which is homoserine • Homo is generally a prefix meaning containing an extra methylene group • This is precursor to homocysteine  methionine • It also leads to threonine homoserine Biochem: AAmetab 2; Nuc 1

  23. Phospho-homoserine Homoserine to threonine • Homoserine phosphorylated with ATP as phosphate donor • Phosphohomoserine dephosphorylated with movement of -OH from one carbon to another: threonine results threonine Biochem: AAmetab 2; Nuc 1

  24. Homoserine to methionine • Three reactions converthomoserine to homocysteine • 5-methyltetrahydrofolate servesas a methyl donor to converthomocysteine to methioninevia methionine synthase • This enzyme exists in humans but its activity is low and [homocysteine] is low; • So methionine is essential in humans homocysteine Biochem: AAmetab 2; Nuc 1

  25. Specifics for lysine 2,3-dihydro-picolinate • Aspartyl semialdehyde condenses with pyruvate to form 2,3-dihydropicolinate • Reduced again to 2,3,4,5-tetrahydropicolinate • Acylated (via AcylCoA) to N-acyl-2-amino-6-oxopimelate • Transaminated to N-acyl-2,6-diaminopimelate • Deacylated to L,L-N-acyl-2,6-diaminopimelate • Epimerase converts that to meso form • That’s decarboxylated to lysine Biochem: AAmetab 2; Nuc 1

  26. AA moles ATP essen- 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 ATP essen- 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 (cf. box 17.3) Biochem: AAmetab 2; Nuc 1

  27. Branched-chain aliphatics:isoleucine and valine -ketobutyrate • Derived from pyruvate or -ketobutyrate • 2 pyruvate -ketoisovalerate + CO2 • pyr + -ketobutyrate -keto--methylvalerate + CO2 • These products are transaminated to ile and val -keto-methylvalerate Biochem: AAmetab 2; Nuc 1

  28. Leucine • Also derived from -ketoisovalerate; • An extra methylene is inserted between the polar end and the isopropyl group • Final reaction is another transamination Biochem: AAmetab 2; Nuc 1

  29. shikimate Aromatics: phe and tyr • Common pathways for phe,tyr,trp via shikimate and chorismate • For phe, tyr: chorismate converted to prephenate • Prephenate can be aromatized with or without a 4-OH group to lead to phe,tyr chorismate Biochem: AAmetab 2; Nuc 1

  30. Reaction specifics prephenate • Prephenate is oxidized and dehydroxylated in two steps to phenylpyruvate • Or it is oxidized to 4-OH-phenylpyruvate • Transaminations of those -ketoacids yield the final amino acids 4-hydroxy-phenyl-pyruvate Biochem: AAmetab 2; Nuc 1

  31. Chorismate mutase • Isomerase, converts chorismate to prephenate • In E.coli: 2 versions depending on which path the product is heading to • Active sites are similar in all organisms but architecture is very different • Catalytic triad similar to serine proteases PDB 1DBF, 1.3Å42 kDa trimerEC 5.4.99.5B.subtilis Biochem: AAmetab 2; Nuc 1

  32. Path to tryptophan:anthranilate synthase • Chorismate reacts with glutamine and is aromatized to anthranilate:chorismate + gln  anthranilate + pyruvate + glutamate PDB 1I1Q157 kDaheterotetramerheterodimershownEC 4.1.3.271.9ÅSalmonella anthranilate Biochem: AAmetab 2; Nuc 1

  33. Anthranilate to indole • Four-step pathway: • phosphoribosyl pyrophosphate (PRPP) contributes a phosphoribosyl group • Sugar ring opens and rearranges • Result is decarboxylated and forms a second ring to form indole 3-glycerinphosphate • Glyceraldehyde-3-P is released to leave indole Biochem: AAmetab 2; Nuc 1

  34. Tryptophan synthase • Indole + ser tryptophan + H2O • PLP-dependent enzyme, but different in how it uses PLP from the transaminases PDB 2CLF146 kDaheterotetramer;heterodimershownEC 4.2.1.201.7ÅSalmonella Biochem: AAmetab 2; Nuc 1

  35. Genetic control of aromatic aa synthesis • In E.coli and many other bacteria, a single operon controls several chorismate-related genes: the aro pathway Biochem: AAmetab 2; Nuc 1

  36. Histidine (fig. 17.22) • Start with PRPP and ATP: form phosphoribosyl ATP • 3 reactions involving glutamine as nitrogen donor for ring lead to imidazole glycerol phosphate • That gets modified and transaminated to make histidine Biochem: AAmetab 2; Nuc 1

  37. What do we do with amino acids? • Obviously a lot of them serve as building-blocks for protein and peptide synthesis via ribosomal mechanisms • Also serve as metabolites, getting converted to other compounds or getting oxidized as fuel Biochem: AAmetab 2; Nuc 1

  38. Transaminations • Generally two stages: • amino acid + -ketoglutarate -keto acid + glutamate • Glutamate + NAD+ + H2O -ketoglutarate + NADH + H+ + NH4+ • Net reaction isamino acid + NAD+ + H2O -keto acid + NADH + H+ + NH4+ Biochem: AAmetab 2; Nuc 1

  39. Glucogenic and ketogenic amino acids • Degradation of many amino acids lead to TCA cycle intermediates or pyruvate • therefore these can be built back up to glucose; • these are called glucogenic • Degradation of others leads to acetyl CoA and related compounds • these cannot be built back up to glucose except via the glyoxalate shuttle • these are called ketogenic Biochem: AAmetab 2; Nuc 1

  40. Glucogenic amino acids • Amino acids that can be catabolized to produce building blocks that lead to glucose without help of glyoxalate pathway • Most produce succinate, succinyl CoA, fumarate, -ketoglutarate, or oxaloacetate Biochem: AAmetab 2; Nuc 1

  41. Ketogenic amino acids • These do not produce TCA cycle intermediates, but rather produce acetyl CoA or its close relatives • Can be built back up into fats or ketone bodies • By convention, amino acids that produce both acetyl CoA and TCA cycle intermediates or pyruvate are considered glucogenic • On that basis, only leu and lys are purely ketogenic Biochem: AAmetab 2; Nuc 1

  42. Serine-based metabolites • Serine is a building block for sphinganine and therefore for sphingolipids • Serine also leads to phosphatidylserine, which is important by itself and can be metabolized to phosphatidylethanolamine and phosphatidylcholine Biochem: AAmetab 2; Nuc 1

  43. Serine degradation • Two paths for degrading serine: • PLP-dependent serine dehydratase simply deaminates ser to pyruvate;this enzyme is like trp synthase • More common: SHMT transfers hydroxymethyl group to THF, leaving glycine; we’ve seen that one as a biosynthetic enzyme for making glycine Serine dehydratase PDB 1P5J41 kDa monomer HumanEC 4.3.1.17, 2.5Å Biochem: AAmetab 2; Nuc 1

  44. Glycine-based metabolites porphobilinogen • Glycine is a source for purines, glyoxylate, creatine phosphate, and (with the help of succinyl CoA) porphobilinogen, whence we get porphyrins, and from those we get chlorophyll, heme, and cobalamin Biochem: AAmetab 2; Nuc 1

  45. Glycine cleavage system • Glycine + H2O + NAD+ + THF NADH + H+ + HCO3- + NH4+ +5,10-methyleneTHF • Complex system: PLP, lipoamide, FAD prosthetic groups • Lipoamide swinging arm works as in pyruvate dehydrogenase T protein(aminomethyl-transferase) of glycine cleavage systemPDB 1V5V93 kDa dimerPyrococcusEC 2.1.2.10, 1.5Å Biochem: AAmetab 2; Nuc 1

  46. asp, glu, ala degradation I • Standard transmination converts aspartate to oxaloacetate with release of glutamate, which then can be deaminated to re-form -ketoglutarate: • asp + -kg  oxaloacetate + glu • glu + NAD+ + H2O -kg + NADH + H+ + NH4+ Biochem: AAmetab 2; Nuc 1

  47. Asp, glu, ala degradation II • Deamination converts glutamateto -ketoglutarate, as above • Standard transamination converts alanine to pyruvate according to the same logic as asp Biochem: AAmetab 2; Nuc 1

  48. All three of these are glucogenic! • -ketoglutarate and oxaloacetate are TCA cycle intermediates • Pyruvate can be regarded as a glucose precursor via the gluconeogenesis pathways (pyr -> OAA -> PEP -> …) Biochem: AAmetab 2; Nuc 1

  49. Degradation of asn, gln • Asparagine and glutamine are deaminated on their side-chains to asp and glu • Thus they lead to oxaloacetate and -ketoglutarate, respectively • So they’re glucogenic • The initial hydrolyses (deaminations) are catalyzed by asparaginase and glutaminase AsparaginasePDB 1O7J144 kDa tetramerEC 3.5.1.1, 1ÅErwinia chrysanthemi Biochem: AAmetab 2; Nuc 1

  50. Arginine degradation ArginasePDB 2AEB 212 kDa hexamer Dimer shown HumanEC 3.5.3.1, 1.29Å • Arginine is hydrolyzed to urea and ornithine as part of the urea cycle; enzyme is arginase • PLP-dependent enzyme converts ornithine to glu -semialdehyde • That’s oxidized to glutamate Biochem: AAmetab 2; Nuc 1

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