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Amino Acid Metabolism II & Nucleic Acid Chemistry I Insights

This lecture covers the complexities of amino acid pathways, including synthesis, degradation, and cellular localization. It also delves into nucleic acid chemistry focusing on N-rings, nucleosides, and more. Detailed enzyme reactions and key metabolites are discussed.

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Amino Acid Metabolism II & Nucleic Acid Chemistry I Insights

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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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