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Nitrogen Metabolism 1

Nitrogen Metabolism 1. Andy Howard Biochemistry Lectures, Spring 2019 Thursday 25 April 2019. Nitrogen metabolism. Amino acid anabolism sometimes involves little more than transamination, but often is more complex Amino acid catabolism feeds the TCA cycle and acetyl CoA. Nitrogenase

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Nitrogen Metabolism 1

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  1. Nitrogen Metabolism 1 Andy HowardBiochemistry Lectures, Spring 2019Thursday 25 April 2019

  2. Nitrogen metabolism • Amino acid anabolism sometimes involves little more than transamination, but often is more complex • Amino acid catabolism feeds the TCA cycle and acetyl CoA Nitrogen Metabolism 1

  3. Nitrogenase Nitrogen cycles AA anabolism Glutamate Transaminations Simple syntheses Complex syntheses AA catabolism Transaminations Glucogenic & Ketogenic aa’s What we’ll discuss Nitrogen Metabolism 1

  4. The nitrogen pool • Nitrogen fixation from air (N2 NH3) doesn’t produce a large percentage of circulating biological nitrogen but it’s the ultimate source of most of it • Other entries in pool: nitrate (NO3 -), nitrite (NO2-) • Most of this difficult biochemistry is bacterial Nitrogen Metabolism 1

  5. Nitrogenase • Enzyme found in Rhizobium,a bacterium that colonizes & livessymbiotically in the root nodules oflegumes and a few other plants • Also in free-living microorganisms like Azotobacter • Energetically expensive but irreversible path to reduction of dinitrogen to ammonia: • N2 + 8H+ + 8e- + 16 ATP 2NH3 + H2 + 16ADP + 16Pi Nitrogen Metabolism 1

  6. Nitrogenase structure • Multi-component complex • Mo-Fe active site in actual N2-fixing component AzotobacterNitrogenaseMo-Fe, Fe proteins350 kDa heterooctamerEC 1.18.6.1PDB 1G20, 2.2Å Nitrogen Metabolism 1

  7. Mechanistic intermediates • Probably proceeds via diimine and hydrazine: • NN + 2e- + 2H+ H-N=N-H • H-N=N-H + 2e- + 2H+ H2N-NH2 • H2N-NH2 + 2e- + 2H+ 2 NH3 • 2e- + 2H+ H2  Nitrogen Metabolism 1

  8. Ammonia, nitrate, nitrite Alcaligines Nitrite Reductase111 kDa trimermonomer shownPDB 2BO0, 1.35Å • Ammonia comes from decayed organisms and is oxidized in soil bacteria to nitrate (nitrification) • Nitrate reductase and nitrite reductase found in plants and microorganisms Nitrogen Metabolism 1

  9. Reductase reactions NO3- + 2e- + 2H+ NO2- + H2O NO2- + 6e- + 7H+ NH3 + 2 H2O Nitrogen Metabolism 1

  10. Essential and non-essential amino acids • An amino acid is defined as essential if it must be obtained within the diet • In general the essential amino acids are the ones that have complicated and highly ATP-dependent biosynthetic pathways • Of course, it depends on the organism Nitrogen Metabolism 1

  11. 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 Nitrogen Metabolism 1

  12. Transaminations • General process of interconverting -amino acids and -ketoacids • Primary way that N gets incorporated into non-N-containing structures Nitrogen Metabolism 1

  13. Reaction dynamics • All (?) transaminations involve PLP as a cofactor: see mechanism in textbook • These are actually oxidation-reduction reactions, since we’re swapping an amine (carbon oxidation state +2) for a carbonyl (carbon oxidation state 0) • But there is no external oxidizing agent E.coli Aspartateaminotransferase EC 2.6.1.1 87 kDa dimer; Monomer shownPDB 2Q7W, 1.4Å Nitrogen Metabolism 1

  14. Examples of transaminases Reactants Products Trans-Keto acid amino acid keto acid amino acid aminase Pyruvate glutamate -k-glutarate alanine pyruvate Pyruvate aspartate oxaloacetate alanine pyruvate Oxaloacetate glutamate -k-glutarate aspartate aspartate 3-phosphono- glutamate -k-glutarate phosphoserine phospo- hydroxypyruvate serine 4-OH-phenyl- glutamate -k-glutarate tyrosine tyrosinepyruvate Nitrogen Metabolism 1

  15. Catabolic or anabolic? • From the point of view of available pools of amino acids, these are amphibolic: • They involve synthesis of one amino acid at the expense of another Nitrogen Metabolism 1

  16. iClicker question #1 1. The distinction between essential and non-essential amino acids relates to • (a) whether an organism can synthesize the amino acid • (b) how we degrade the amino acid • (c) how many kJ/mol can be derived from its catabolism • (d) how many sulfurs are present Nitrogen Metabolism 1

  17. Some are complex and energy-requiring Can be logically divided according to chemical properties of the target amino acids: Small Branched-chain aliphatic Neutral polar Acidic Basic Aromatic Sulfur-containing Biosynthetic pathways to specific amino acids Nitrogen Metabolism 1

  18. Which amino acids in which categories? Category Amino acids • Small gly, ala, ?pro? • Branched-chain val, leu, ile • Neutral polar asn, gln, ser, thr • Acidic asp, glu • Basic lys, arg • Aromatic phe, tyr, trp, his • Sulfur-containing cys, met Nitrogen Metabolism 1

  19. 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: Glu dehydrogenase PDB 1BGV296 kDa hexamermonomer shown EC 1.4.1.2, 1.9ÅClostridium Nitrogen Metabolism 1

  20. Glutamate dehydrogenase reaction -ketoglutarate + NH4+ + NAD(P)H + H+NAD(P)+ + H2O + glutamate Nitrogen Metabolism 1

  21. 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 Human Glutamine synthetase EC 6.3.1.2211 kDa pentamerPDB 2OJW, 2.05Å Nitrogen Metabolism 1

  22. Aspartate and asparagine • Asp is simple:transamination of oxaloacetate • Asn is straightforward too • asparagine synthetase moves amine from gln to asp, leaving glu (another ligase) • Gln + asp + ATP  AMP + PPi + glu + asn E.coli Asparagine synthetase B EC 6.3.5.4243 kDa tetramer PDB 1CT9, 2Å Nitrogen Metabolism 1

  23. 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 Human Phosphoserine phosphatase49 kDa dimerEC 3.1.3.3PDB 1NNL,1.53Å Nitrogen Metabolism 1

  24. 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 Thermus thermophilus SHMT90 kDa dimerEC 2.1.2.1 PDB 2DKJ,1.15Å Nitrogen Metabolism 1

  25. Arginine & proline Glutamate semialdehyde • Two routes: • Glutamate to glutamate semialdehyde • that cyclizes to 1-pyrroline 5-carboxylateand thence to proline • Glutamate semialdehyde can alsobe converted to ornithine and thence to arg • Alternative: glutamate acetylatedto N-acetyl-glutamate-5-semialdehydeand thence to ornithine ornithine Nitrogen Metabolism 1

  26. Glutamate to P5C • Single enzyme can interconvert glutamate and 1-pyrroline carboxylate:1-pyrroline-5-carboxylate dehydrogenase • 3-layer  sandwich protein Thermus thermophilus PCD300 kDa hexamerdimer shownEC 1.5.1.12 PDB 2BJA, 1.9Å Nitrogen Metabolism 1

  27. Pyrroline-5-carboxylate to proline • Pyrroline-5-carboxylate reduced to proline • Large, NAD(P)H-dependent enzyme Human Pyrroline-5-carboxylate reductaseEC 1.5.1.2354 kDa decamerpentamer shownPDB 2IZZ, 1.95Å Nitrogen Metabolism 1

  28. 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+ + Pi Thermatoga maritima -glutamyl phosphate reductase47 kDa monomerEC 1.2.1.41PDB 1O20, 2Å Glu-5-P Nitrogen Metabolism 1

  29. Glu semialdehyde to ornithine • This is just another transamination, catalyzed by ornithine aminotransferase:glu-5-semialdehyde + glu/asp  ornithine + -ketoglutarate / oxaloacetate • Typical PLP-dependent reaction Human OAT193 kDa tetramerEC 2.6.1.13PDB 2OAT, 1.95Å ornithine Nitrogen Metabolism 1

  30. Carbamoyl phosphate Ornithine to citrulline • Ornithine condenses with carbamoyl phosphate to form citrulline with the help of ornithine transcarbamoylase 110 kDa trimerE.coliEC 2.1.3.3, PDB 1DUV, 1.7Å citrulline Nitrogen Metabolism 1

  31. Citrulline to argininosuccinate • Citrulline condenses with aspartate using ATP hydrolysis to drive it forward to L-argininosuccinate:citrulline + aspartate + ATP L-argininosuccinate + AMP + PPi Argininosuccinate synthasePDB 2NZ2, 2.4ÅEC 6.3.4.5 200 kDa tetramermonomer shown Nitrogen Metabolism 1

  32. Argininosuccinate to arginine • Fumarate extracted,leaving arginine • Argininosuccinate lyase is also -crystallin, one of the moonlighting proteins: it’s a component of eye lenses E.coli ASL 100 kDa dimerEC 4.3.2.1, 2.44ÅPDB 1TJ7, 2.44 fumarate Nitrogen Metabolism 1

  33. 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. Nitrogen Metabolism 1

  34. Cysteine synthesis in plants and bacteria • serine + Acetyl CoA O-acetylserine + HSCoA • O-acetylserine + S2- + H+ cysteine + acetate • Ser acetyltransferase is inhibited by cysteine Haemophilus serine acetyltransferaseEC 2.3.1.30176 kDa hexamerdimer shownPDB 1SSQ, 1.85Å O-acetylserine Nitrogen Metabolism 1

  35. Animal pathway to cys • Ser + homocysteine (from met) fuse to form cystathionine + H2O • Cystathionine + H2O NH4+ + cysteine + -ketobutyrate Yeast Cystathionine -lyaseEC 4.4.1.1PDB 1N8P, 2.6 Å 173 kDa tetramer cystathionine Nitrogen Metabolism 1

  36. Amino acids we’ve already covered Acids and amides:glu, gln, asp, asn Simple:ala, ser, gly Others: arg, pro, cys Essential but straightforward met, thr val, leu, ile Essential & Ugly:lys, phe, tyr, trp, his Marching through the list of twenty amino acids Nitrogen Metabolism 1

  37. 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 ArabidopsisAspartate kinase112 kDa dimerEC 2.7.2.4PDB 2CDQ, 2.85Å Nitrogen Metabolism 1

  38. 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 Nitrogen Metabolism 1

  39. 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 Nitrogen Metabolism 1

  40. homocysteine Homoserine to met • 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 methionine Nitrogen Metabolism 1

  41. 2,3-dihydro-picolinate Lysine I 2,3,4,5-tetrahydro-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 N-succinyl-2-amino-6-oxopimelate Nitrogen Metabolism 1

  42. Lysine II • N-acyl-2-amino-6-oxopimelate transaminated to N-acyl-2,6-diaminopimelate • Deacylated to L,L-2,6-diaminopimelate • Epimerase converts that to meso form • That’s decarboxylated to lysine Nitrogen Metabolism 1

  43. What’s left? (= “done”) 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 Nitrogen Metabolism 1

  44. 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 val and ile α-keto-isovalerate -keto-methylvalerate Nitrogen Metabolism 1

  45. Leucine -ketoisovalerate • Also derived from -ketoisovalerate; • An extra methylene is inserted between the polar end and the isopropyl group • Final reaction is another transamination Nitrogen Metabolism 1

  46. 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 Nitrogen Metabolism 1

  47. prephenate Reaction specifics • 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 Nitrogen Metabolism 1

  48. 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 B.subtilis chorismate mutase42 kDa trimerEC 5.4.99.5 PDB 1DBF, 1.3Å Nitrogen Metabolism 1

  49. PRPP Histidine • 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 imidazole glycerol phosphate Nitrogen Metabolism 1

  50. anthranilate Tryptophan Also via chorismate Sidechain amide transferred to chorismate Conversion to anthranilate PRPP provides phosphoribosyl moiety; this sets up indole glycerol phosphate Tryptophan synthase eliminates glyc-P, adds ser Nitrogen Metabolism 1

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