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Nitrogen Metabolism: An Overview of Amino Acid Anabolism and Catabolism

Understand the complex processes of amino acid synthesis and breakdown, including transaminations and nitrogen fixation. Learn about essential and non-essential amino acids and the biochemical pathways involved.

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Nitrogen Metabolism: An Overview of Amino Acid Anabolism and Catabolism

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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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