Overview of Nitrogen Metabolism and Biosynthesis of Amino Acids CH353 January 22, 2008

The Nitrogen Cycle Nitrogen Fixation Nitrification Denitrification N2 → NH4+ Nitrification NH3 → NO2- → NO3- Denitrification NO2- & NO3- → N2 Nitrogen Assimilation NH4+ → Organic nitrogen Deamination Organic nitrogen → NH4+

N2 + 10H+ + 8e- + 16ATP → 2NH4+ + 16ADP + 16Pi + H2 Nitrogen Fixation Nitrogen Reduction Reaction N2 + 3 H2 → 2NH3 ΔG′º = -33.5 kJ/mol Exergonic reaction High activation energy Stability of N2 triple bond D = 930 kJ/mol Biological Nitrogen Reduction N2 + 10H+ + 8e- + 16ATP → 2NH4+ + 16ADP + 16Pi + H2

Nitrogenase Complex Dinitrogenase reductase Dinitrogenase Dimer (Mr 60,000) of same subunits One 4Fe-4S between subunits One ATP/ADP site per subunit ATP binding enhances reduction potential: E′º -300 → -420 mV Dinitrogenase Tetramer (Mr 240,000): 2 copies of 2 different subunits 2 Mo, 32 Fe, 30 S per tetramer 8 dinitrogenase reductase dimers each transfers one e- to dinitrogenase

Nitrogen Assimilation Glutamine Synthetase Glutamate + NH4+ + ATP → Glutamine + ADP + Pi + H+ Glutamate Synthase (plants, bacteria) α-Ketoglutarate + Glutamine + NADPH + H+ → 2 Glutamate + NADP+ Net Reaction: α-Ketoglutarate + NH4+ + NADPH + ATP → Glutamate + NADP+ + ADP + Pi Glutamate Dehydrogenase α-Ketoglutarate + NH4+ + NADPH → Glutamate + NADP+

Regulation of Glutamine Synthetase (E. coli) Covalent Modification Adenylylation inactivates glutamine synthase Adenylyltransferase (AT) regulated by PII Uridylylation of PII determines regulation: no UMP - adenylylation UMP – deadenylylation α-Ketoglutarate & ATP stimulate uridylylation ↑ Glutamine synthesis Glutamine & Pi inhibit uridylylation ↓ Glutamine synthesis

Regulation of Glutamine Synthetase (E. coli) Transcription Activation Uridylylated PII activates gene encoding glutamine synthetase Allosteric Inhibition Glutamine synthetase is inhibited by alanine, glycine and products of glutamine metabolism Binding of each metabolite causes partial inhibition; binding of all completely inhibits enzyme Overall effect of 8 inhibitors is more than additive: example of Concerted Inhibition

Biosynthetic Reactions with Glutamine Amidotransferase reactions Glutamine is hydrolyzed to glutamate + NH3 within the enzyme Acceptor hydroxyl or ketone often activated with ATP Glutamine + Aspartate + ATP ↓ Glutamate + Asparagine + AMP + PPi

Overview of Amino Acid Biosynthesis Ribose 5-phosphate Histidine 3-Phosphoglycerate Serine Glycine Cysteine Erythrose-4-phosphate Phosphoenolpyruvate Tryptophan Phenylalanine Tyrosine Pyruvate Alanine Valine Isoleucine Leucine Oxaloacetate Aspartate Asparagine Methionine Lysine Threonine α-Ketoglutarate Glutamate Glutamine Proline Arginine Key: nonessential conditional essential

Reactions with Pyridoxal Phosphate Transamination (aminotransferase) reactions Glutamate + Pyruvate α-Ketoglutarate + Alanine Glutamate + Oxaloacetate α-Ketoglutarate + Aspartate

Amino Acids from α-Ketoglutarate

Amino Acids from α-Ketoglutarate Glutamate Glutamine Glutamate g-semialdehyde Arginine Proline Ornithine Urea Cycle

Biosynthesis of Proline and Arginine ornithine aminotransferase Glutamate α-Ketoglutarate

Amino Acids from 3-Phosphoglycerate

Biosynthesis of Serine and Glycine

Cofactors for One-Carbon Metabolism

One-Carbon Units on Tetrahydrofolate tetrahydrofolate (H4 folate) is derived from folic acid one-carbons on H4 folate can have 3 redox states hydroxymethyl group of serine and formate are primary entry metabolites reversible conversions except for production of N 5-methyl H4 folate

The Activated Methyl Cycle S-Adenosyl methionine is the methyl donor for nearly all reactions Methyl group of methionine is replenished with N5-methyl H4 folate Methionine synthase requires coenzyme B12 (from vitamin B12)

Biosynthesis of Cysteine Only bacteria and plants can assimilate inorganic sulfur In mammals, cysteine is conditionally essential deriving its sulfur from methionine Cysteine is biosynthesized from serine and homocysteine, a methionine metabolite Sulfhydryl is transferred from homocysteine to serine in two PLP dependent steps with cystathionine as intermediate

Study Problem High serum homocysteine level is a risk factor for coronary heart disease and arteriosclerosis Some individuals with high homocysteine have variations in the cystathionine β-synthase gene A combination of vitamin supplements is recommended for alleviating high homocysteine Based upon the pathways for homocysteine synthesis and utilization, which 3 vitamins would you recommend?

Amino Acids from Oxaloacetate and Pyruvate

Amino Acids from Oxaloacetate Aspartate Asparagine Pyruvate Aspartate β-semialdehyde Dihydropicolinate Homoserine Lysine Methionine Threonine

Biosynthesis of Lysine, Methione & Threonine Dihydropicolinate Lysine Methionine & Threonine

Biosynthesis of Methionine

Biosynthesis of Methionine

Biosynthesis of Cysteine Biosynthesis of Methionine Homocysteine Serine Cysteine Homoserine H2O cystathionine β-synthase cystathionine γ-synthase γ β PLP Cystathionine H2O cystathionine β-lyase cystathionine γ-lyase PLP α-Ketobutyrate Cysteine Pyruvate Homocysteine

Biosynthesis of Threonine

Amino Acids from Pyruvate Threonine Pyruvate Alanine CO2 α-Ketobutyrate acetolactate synthetase Pyruvate Acetyl-CoA Same enzymes for both valine and isoleucine CO2 Isoleucine Valine Leucine

Biosynthesis of Isoleucine and Valine Valine & Leucine

Regulation of Amino Acid Biosynthesis Multiple Isozymes for Aspartokinase (A1, A2, A3) Homoserine dehydrogenase (B1, B2) Threonine dehydratase (C1, C2) Allosteric regulation of selective isozymes – some unregulated Sequential feedback inhibition Same product inhibits its biosynthetic path at multiple sites Inhibits first enzyme in pathway

Biosynthesis of Aromatic Amino Acids

Biosynthesis of Aromatic Amino Acids Phosphoenolpyruvate Erythrose 4-phosphate Shikimate Pyruvate Glu Gln Chorismate Anthranilate PRPP Prephenate CO2 CO2 Serine CO2 Tyrosine Phenylalanine Glyceraldehyde 3-phosphate Tryptophan

Conversion of Phenylalanine to Tyrosine Tyrosine is a conditionally essential amino acid It can be synthesized from phenylalanine as part of its catabolic pathway

Biosynthesis of Histidine PRPP ATP Glutamine Purine Biosynthesis AICAR Glutamate Histidine