Nitrogen AssimilationSlide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Rules of EngagementSlide 12Slide 13Nitrogen Assimilation•How is NH3 incorporated into organic molecules?•Glutamate Dehydrogenase vs Glutamate Synthase•Properties of Glutamine Synthetase•Regulation of Glutamine Synthetase•Glutamine as a major nitrogen donorNH3Glutamine-Ketoglutarate GlutamateCO2 ATPAspartateGlutamateAsparagineCarbamoylphosphate Arginine UreaOtheraminoacids Purine nucleotides,Cytidine nucleotides Amino sugars,Tryptophan, Histidine Pyrimidine nucleotides 3 Gateways to Biological Molecules3 Gateways to Biological MoleculesOf the 3, the most versatile is glutamineNoneThe only inorganicnitrogen source formammals is NH3Glutamate Dehydrogenase-Kg + NH3 + NAD(P)H + 2H+Glutamate + NAD(P)++ H2OBacteria and PlantsMake glutamate, assimilate NH3AnimalsProvide NH3 to urea cycle, provide -Kg to KrebsMitochondrial location(-) by ATP, GTP(+) by ADP, GDPGlutamate is major solute in thebacteriaHigh Km for NH3 limits forwardGlutamate Synthase (Bacteria Only)-Kg + glutamine + NADPH + H+2 glutamates + NADP+Glutamine is the nitrogen donorReaction is a reductive aminationGlutamine Synthetase is a Primary RegulatoryPoint in Nitrogen MetabolismProperties of Bacterial Enzyme12 identical 50,000 mwt subunitsCombined Mwt of 600,000Hexagonal stacked rings66Each subunits subject to allostericregulation8 allosteric sites on each subunitTOP VIEWSIDE VIEWOne covalent site (Tyr 397)Regulation is cumulativeGlutamine SynthetaseGlutamate + NH4+ + ATP Glutamine + ADP + Pi (Biosynthesis - anabolic)-Ketoglutarate(Degradation – catabolic)Take Home: Shutting down the enzyme favors using glutamate as an energy substrate. Not shutting down the enzyme keeps the cell in a biosynthetic mode.OPOOOOCH2OHOHAdenineAdenylylated tyrosine residueEnzymeEnzymeTyrTyrNH3+ATP+COOCH2CH2C NH3COOH+CH2CH2C NH3COOH+CONH2+ ADP + PiCovalent Modification by AdenylylationAllosteric EffectorsEach inhibits Glutamine Synthetase (Favors boosting cell energyor shutting down a pathway requiring glutamine )AMPCTP(Low energy state exists, oxidize -Kg)(End product of pyrimidine synthesis)HistidineGlycineTryptophanAlanineAmino acids that are allosteric effectorsGlucosamine (Glutamine sufficient for amino sugar synthesis)Carbamoyl-PO4(Glutamine sufficient for pyrimidine synthesis)Covalent RegulationAdenylylation of Tyrosine 397 on EACH of the 12 subunitsAdenyl = group attachedAdenylylation = group attached and processFor example:An acyl group attached via an acylation reaction isACYLACYLATIONORACYLYLATION(PRONOUNCED ACIL-LIL-ATION)2 TransferasesEach puts on and takes off groupsAT (Adenylyltransferase - adenylylates GS)UT (Uridylyltransferase - uridylylates PII)One Regulatory ProteinPIITwo StatesAT-PIIAdenylatesAT-PII-UMPDeadenylylatesCovalent Regulation of Glutamine SynthetaseRules of Engagement•Transferases catalyze adenylylation (uridylylation) and deadenylylation (deuridylylation reactions•Adenylylation shuts GS down cumulatively Deadenylylation turns GS back on cumulatively•AT requires PII to adenylylate•AT requires PII-UMP to deadenylylateGS*ATPGSAMPAT= UselessATPII= AdenylylatesAMPPIIUMPPIIAT PIIATPII UMPAT PIIUMP= DeadenylylatesUT = Uridylylates UTPPPiUMPActiveLess ActiveUT URUR= Deuridylylates(2 activities onsame enzyme)Uridylate removing EnzymePIIUMPPIIUTP PPiUMP(-) glutamine(+) -KgUMPPIIPIIActivates GSInactivates GSATPDeadenylylatesLeads to adenylylationURUTUTURResponsive to cell’snitrogen