Andy Howard Biochemistry Lectures, Spring 2019 Thursday 25 April 2019 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 04/25/2019 Nitrogen Metabolism 1

What we’ll discuss Nitrogenase Nitrogen cycles AA anabolism Glutamate Transaminations Simple syntheses Complex syntheses AA catabolism Transaminations Glucogenic & Ketogenic aa’s 04/25/2019 Nitrogen Metabolism 1

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 04/25/2019 Nitrogen Metabolism 1

Nitrogenase Enzyme found in Rhizobium, a bacterium that colonizes & lives symbiotically in the root nodules of legumes 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 04/25/2019 Nitrogen Metabolism 1

Nitrogenase structure Multi-component complex Mo-Fe active site in actual N2-fixing component Azotobacter Nitrogenase Mo-Fe, Fe proteins 350 kDa heterooctamer EC 1.18.6.1 PDB 1G20, 2.2Å 04/25/2019 Nitrogen Metabolism 1

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  04/25/2019 Nitrogen Metabolism 1

Ammonia, nitrate, nitrite Ammonia comes from decayed organisms and is oxidized in soil bacteria to nitrate (nitrification) Nitrate reductase and nitrite reductase found in plants and microorganisms Alcaligines Nitrite Reductase 111 kDa trimer monomer shown PDB 2BO0, 1.35Å 04/25/2019 Nitrogen Metabolism 1

Reductase reactions NO3- + 2e- + 2H+  NO2- + H2O NO2- + 6e- + 7H+  NH3 + 2 H2O 04/25/2019 Nitrogen Metabolism 1

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 04/25/2019 Nitrogen Metabolism 1

Essential & non-essential aa’s 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 04/25/2019 Nitrogen Metabolism 1

Transaminations General process of interconverting -amino acids and -ketoacids Primary way that N gets incorporated into non-N-containing structures 04/25/2019 Nitrogen Metabolism 1

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 Aspartate aminotransferase EC 2.6.1.1 87 kDa dimer; Monomer shown PDB 2Q7W, 1.4Å 04/25/2019 Nitrogen Metabolism 1

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 tyrosine pyruvate 04/25/2019 Nitrogen Metabolism 1

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 04/25/2019 Nitrogen Metabolism 1

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 04/25/2019 Nitrogen Metabolism 1

Biosynthetic pathways to specific amino acids 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 04/25/2019 Nitrogen Metabolism 1

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 04/25/2019 Nitrogen Metabolism 1

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 1BGV 296 kDa hexamer monomer shown EC 1.4.1.2, 1.9Å Clostridium 04/25/2019 Nitrogen Metabolism 1

Glutamate dehydrogenase reaction -ketoglutarate + NH4+ + NAD(P)H + H+  NAD(P)+ + H2O + glutamate 04/25/2019 Nitrogen Metabolism 1

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 6.3.1.2 211 kDa pentamer PDB 2OJW, 2.05Å 04/25/2019 Nitrogen Metabolism 1

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 6.3.5.4 243 kDa tetramer PDB 1CT9, 2Å 04/25/2019 Nitrogen Metabolism 1

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 phosphatase 49 kDa dimer EC 3.1.3.3 PDB 1NNL,1.53Å 04/25/2019 Nitrogen Metabolism 1

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 SHMT 90 kDa dimer EC 2.1.2.1 PDB 2DKJ,1.15Å 04/25/2019 Nitrogen Metabolism 1

Arginine & proline Two routes: Glutamate to glutamate semialdehyde that cyclizes to 1-pyrroline 5-carboxylate and 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 04/25/2019 Nitrogen Metabolism 1

Glutamate to P5C Single enzyme can interconvert glutamate and 1-pyrroline carboxylate: 1-pyrroline-5-carboxylate dehydrogenase 3-layer  sandwich protein Thermus thermophilus PCD 300 kDa hexamer dimer shown EC 1.5.1.12 PDB 2BJA, 1.9Å 04/25/2019 Nitrogen Metabolism 1

Pyrroline-5-carboxylate to proline Pyrroline-5-carboxylate reduced to proline Large, NAD(P)H-dependent enzyme Human Pyrroline-5-carboxylate reductase EC 1.5.1.2 354 kDa decamer pentamer shown PDB 2IZZ, 1.95Å 04/25/2019 Nitrogen Metabolism 1

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 reductase 47 kDa monomer EC 1.2.1.41 PDB 1O20, 2Å Glu-5-P 04/25/2019 Nitrogen Metabolism 1

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 OAT 193 kDa tetramer EC 2.6.1.13 PDB 2OAT, 1.95Å ornithine 04/25/2019 Nitrogen Metabolism 1

Ornithine to citrulline Carbamoyl phosphate Ornithine to citrulline Ornithine condenses with carbamoyl phosphate to form citrulline with the help of ornithine transcarbamoylase 110 kDa trimer E.coli EC 2.1.3.3, PDB 1DUV, 1.7Å citrulline 04/25/2019 Nitrogen Metabolism 1

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 synthase PDB 2NZ2, 2.4Å EC 6.3.4.5 200 kDa tetramer monomer shown 04/25/2019 Nitrogen Metabolism 1

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 dimer EC 4.3.2.1, 2.44Å PDB 1TJ7, 2.44 fumarate 04/25/2019 Nitrogen Metabolism 1

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. 04/25/2019 Nitrogen Metabolism 1

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 acetyltransferase EC 2.3.1.30 176 kDa hexamer dimer shown PDB 1SSQ, 1.85Å O-acetylserine 04/25/2019 Nitrogen Metabolism 1

Animal pathway to cys Ser + homocysteine (from met) fuse to form cystathionine + H2O Cystathionine + H2O  NH4+ + cysteine + -ketobutyrate Yeast Cystathionine -lyase EC 4.4.1.1 PDB 1N8P, 2.6 Å 173 kDa tetramer cystathionine 04/25/2019 Nitrogen Metabolism 1

Marching through the list of twenty amino acids 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 04/25/2019 Nitrogen Metabolism 1

Lys, met, thr asp gets phosphorylated and becomes a source for all of these: asp + ATP -aspartyl phosphate + ADP via 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 Arabidopsis Aspartate kinase 112 kDa dimer EC 2.7.2.4 PDB 2CDQ, 2.85Å 04/25/2019 Nitrogen Metabolism 1

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 04/25/2019 Nitrogen Metabolism 1

Homoserine to threonine 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 04/25/2019 Nitrogen Metabolism 1

Homoserine to met Three reactions convert homoserine to homocysteine 5-methyltetrahydrofolate serves as a methyl donor to convert homocysteine to methionine via methionine synthase This enzyme exists in humans but its activity is low and [homocysteine] is low; So methionine is essential in humans methionine 04/25/2019 Nitrogen Metabolism 1

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 04/25/2019 Nitrogen Metabolism 1

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 04/25/2019 Nitrogen Metabolism 1

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 04/25/2019 Nitrogen Metabolism 1

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 04/25/2019 Nitrogen Metabolism 1

Leucine Also derived from -ketoisovalerate; An extra methylene is inserted between the polar end and the isopropyl group Final reaction is another transamination 04/25/2019 Nitrogen Metabolism 1

Aromatics: phe and tyr shikimate 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 04/25/2019 Nitrogen Metabolism 1

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 04/25/2019 Nitrogen Metabolism 1

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 mutase 42 kDa trimer EC 5.4.99.5 PDB 1DBF, 1.3Å 04/25/2019 Nitrogen Metabolism 1

Histidine PRPP 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 04/25/2019 Nitrogen Metabolism 1

Tryptophan Also via chorismate anthranilate 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 04/25/2019 Nitrogen Metabolism 1

iClicker question #2 2. Which of these amino acids is not synthesized through a pathway involving chorismate? (a) phe (b) his (c) trp (d) tyr (e) all of those involve chorismate 04/25/2019 Nitrogen Metabolism 1

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 (including nucleic acids) or getting oxidized as fuel 04/25/2019 Nitrogen Metabolism 1

Transaminations Generally two stages: amino acid + -ketoglutarate  -keto acid + glutamate Glutamate + NAD+ + H2O  -ketoglutarate + NADH + H+ + NH4+ Net reaction is amino acid + NAD+ + H2O  -keto acid- + NADH + H+ + NH4+ 04/25/2019 Nitrogen Metabolism 1

Glucogenic amino acids Degradation of many amino acids lead to TCA cycle intermediates or pyruvate These can be built back up to glucose; So these are called glucogenic 04/25/2019 Nitrogen Metabolism 1

Ketogenic amino acids 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 04/25/2019 Nitrogen Metabolism 1

Glucogenic amino acids Amino acids that can be catabolized to produce building blocks that lead to glucose without help of glyoxalate pathway Most produce pyruvate, -ketoglutarate, succinyl CoA, succinate, fumarate, or oxaloacetate By convention, amino acids that produce both TCA-cycle intermediates and acetyl CoA are labeled as glucogenic 04/25/2019 Nitrogen Metabolism 1