Overview of Nitrogen Metabolismand Biosynthesis of Amino AcidsCH353 January 22, 2008

The Nitrogen CycleNitrogen FixationN2 NH4+NitrificationNH3 NO2- NO3-DenitrificationNO2- & NO3- N2Nitrogen Assimilation NH4+ Organic nitrogenDeaminationOrganic nitrogen NH4+

Nitrogen FixationNitrogen Reduction ReactionN2 + 3 H2 2NH3G = -33.5 kJ/mol Exergonic reactionHigh activation energyStability of N2 triple bond D = 930 kJ/mol Biological Nitrogen ReductionN2 + 10H+ + 8e- + 16ATP 2NH4+ + 16ADP + 16Pi + H2

Nitrogenase ComplexDinitrogenase reductaseDimer (Mr 60,000) of same subunitsOne 4Fe-4S between subunitsOne ATP/ADP site per subunitATP binding enhances reduction potential: E -300 -420 mVDinitrogenaseTetramer (Mr 240,000): 2 copies of 2 different subunits2 Mo, 32 Fe, 30 S per tetramer8 dinitrogenase reductase dimers each transfers one e- to dinitrogenase

Nitrogen AssimilationGlutamine SynthetaseGlutamate + NH4+ + ATP Glutamine + ADP + Pi + H+Glutamate Synthase (plants, bacteria)-Ketoglutarate + Glutamine + NADPH + H+ 2 Glutamate + NADP+Net Reaction:-Ketoglutarate + NH4+ + NADPH + ATP Glutamate + NADP+ + ADP + Pi

Glutamate Dehydrogenase-Ketoglutarate + NH4+ + NADPH Glutamate + NADP+

Regulation of Glutamine Synthetase (E. coli)Covalent ModificationAdenylylation inactivatesglutamine synthaseAdenylyltransferase(AT) regulated by PIIUridylylation of PIIdetermines regulation:no UMP - adenylylationUMP deadenylylation-Ketoglutarate & ATPstimulate uridylylation Glutamine synthesisGlutamine & Piinhibit uridylylation Glutamine synthesis

Regulation of Glutamine Synthetase (E. coli)Transcription ActivationUridylylated PII activates gene encoding glutamine synthetaseAllosteric InhibitionGlutamine synthetase is inhibited by alanine, glycine and products of glutamine metabolismBinding of each metabolite causes partial inhibition; binding of all completely inhibits enzymeOverall effect of 8 inhibitors is more than additive: example of Concerted Inhibition

Biosynthetic Reactions with GlutamineAmidotransferase reactionsGlutamine is hydrolyzed to glutamate + NH3 within the enzymeAcceptor hydroxyl or ketone often activated with ATP

Glutamine + Aspartate + ATPGlutamate + Asparagine + AMP + PPi

Overview of Amino Acid Biosynthesis3-Phosphoglycerate Serine Glycine CysteinePyruvate Alanine Valine Isoleucine Leucine-Ketoglutarate Glutamate Glutamine Proline ArginineRibose 5-phosphate HistidineErythrose-4-phosphatePhosphoenolpyruvate Tryptophan Phenylalanine TyrosineOxaloacetate Aspartate Asparagine Methionine Lysine ThreoninenonessentialconditionalessentialKey:

Reactions with Pyridoxal PhosphateTransamination (aminotransferase) reactionsGlutamate + Pyruvate -Ketoglutarate + Alanine Glutamate + Oxaloacetate -Ketoglutarate + Aspartate

Amino Acids from -Ketoglutarate

Amino Acids from -Ketoglutarate

Biosynthesis of Proline and Arginine

Amino Acids from 3-Phosphoglycerate

Biosynthesis of Serine and Glycine

Cofactors for One-Carbon Metabolism

One-Carbon Units on Tetrahydrofolatetetrahydrofolate (H4 folate) is derived from folic acidone-carbons on H4 folate can have 3 redox stateshydroxymethyl group of serine and formate are primary entry metabolitesreversible conversions except for production of N 5-methyl H4 folate

The Activated Methyl CycleS-Adenosyl methionine is the methyl donor for nearly all reactionsMethyl group of methionine is replenished with N5-methyl H4 folateMethionine synthase requires coenzyme B12 (from vitamin B12)

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

Study ProblemHigh serum homocysteine level is a risk factor for coronary heart disease and arteriosclerosisSome individuals with high homocysteine have variations in the cystathionine -synthase geneA combination of vitamin supplements is recommended for alleviating high homocysteineBased upon the pathways for homocysteine synthesis and utilization, which 3 vitamins would you recommend?

Amino Acids from Oxaloacetate and Pyruvate

Amino Acids from Oxaloacetate Oxaloacetate Aspartate Asparagine Pyruvate Lysine Aspartate -semialdehyde Threonine Methionine HomoserineDihydropicolinate

Biosynthesis of Lysine, Methione & ThreonineLysineMethionine & ThreonineDihydropicolinate

Biosynthesis of Methionine

Biosynthesis of Methionine

HomocysteineSerineCysteineHomoserine-KetobutyrateCysteinePyruvateHomocysteineBiosynthesis of CysteineBiosynthesis of Methionine

Biosynthesis of Threonine

Amino Acids from Pyruvate Alanine Valine Leucine Isoleucine Pyruvate Pyruvate CO2 -Ketobutyrate Threonine acetolactatesynthetase Acetyl-CoA CO2Same enzymes for both valine and isoleucine

Biosynthesis of Isoleucine and ValineIsoleucineValine & Leucine

Regulation of Amino Acid BiosynthesisMultiple Isozymes forAspartokinase (A1, A2, A3)Homoserine dehydrogenase (B1, B2)Threonine dehydratase (C1, C2)Allosteric regulation of selective isozymes some unregulatedSequential feedback inhibitionSame product inhibits its biosynthetic path at multiple sitesInhibits first enzyme in pathway

Biosynthesis of Aromatic Amino Acids

Biosynthesis of Aromatic Amino Acids Phosphoenolpyruvate Erythrose 4-phosphate ShikimateChorismatePrephenateCO2CO2 Tyrosine Phenylalanine AnthranilatePyruvate PRPP CO2 Serine Glyceraldehyde 3-phosphate Tryptophan Glu Gln

Conversion of Phenylalanine to TyrosineTyrosine is a conditionally essential amino acidIt can be synthesized from phenylalanine as part of its catabolic pathway

Biosynthesis of Histidine PRPP ATP Histidine AICARPurineBiosynthesisGlutamineGlutamate