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Nitrogen Assimilation

Nitrogen Assimilation. How is NH 3 incorporated into organic molecules? Glutamate Dehydrogenase vs Glutamate Synthase Properties of Glutamine Synthetase Regulation of Glutamine Synthetase Glutamine as a major nitrogen donor. CO 2. ATP. NH 3. Carbamoyl phosphate. Glutamate.

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Nitrogen Assimilation

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  1. Nitrogen 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 donor

  2. CO2 ATP NH3 Carbamoyl phosphate Glutamate Glutamine Asparagine None Other amino acids Purine nucleotides, Cytidine nucleotides Amino sugars, Tryptophan, Histidine Pyrimidine nucleotides Arginine Urea The only inorganic nitrogen source for mammals is NH3 3 Gateways to Biological Molecules a-Ketoglutarate Aspartate Glutamate Of the 3, the most versatile is glutamine

  3. Glutamate Dehydrogenase -Kg + NH3 + NAD(P)H + 2H+ Glutamate + NAD(P)+ + H2O Bacteria and Plants Make glutamate, assimilate NH3 Glutamate is major solute in the bacteria Provide NH3 to urea cycle, provide -Kg to Krebs Animals High Km for NH3 limits forward Mitochondrial location (-) by ATP, GTP (+) by ADP, GDP

  4. Glutamate Synthase (Bacteria Only) -Kg + glutamine + NADPH + H+ 2 glutamates + NADP+ Glutamine is the nitrogen donor Reaction is a reductive amination

  5. Each subunits subject to allosteric regulation Glutamine Synthetase is a Primary Regulatory Point in Nitrogen Metabolism Properties of Bacterial Enzyme 12 identical 50,000 mwt subunits 6 Combined Mwt of 600,000 6 Hexagonal stacked rings SIDE VIEW 8 allosteric sites on each subunit One covalent site (Tyr 397) TOP VIEW Regulation is cumulative

  6. Glutamate + NH4+ + ATP Glutamine + ADP + Pi Glutamine Synthetase (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.

  7. Adenylylated tyrosine residue O Enzyme O C H P O 2 A d e n i n e O O Tyr O H O H Covalent Modification by Adenylylation O C N H C O O 2 C H C H 2 2 C H C H 2 2 + + H C N H H C N H + A D P + P + N H + A T P i 3 3 3 C O O C O O

  8. Allosteric Effectors Each inhibits Glutamine Synthetase (Favors boosting cell energy or shutting down a pathway requiring glutamine ) AMP (Low energy state exists, oxidize -Kg) CTP (End product of pyrimidine synthesis) Histidine Glycine Amino acids that are allosteric effectors Tryptophan Alanine Glucosamine (Glutamine sufficient for amino sugar synthesis) (Glutamine sufficient for pyrimidine synthesis) Carbamoyl-PO4

  9. Covalent Regulation Adenylylation of Tyrosine 397 on EACH of the 12 subunits Adenyl = group attached Adenylylation = group attached and process For example: An acyl group attached via an acylation reaction is ACYLACYLATION OR ACYLYLATION (PRONOUNCED ACIL-LIL-ATION)

  10. Covalent Regulation of Glutamine Synthetase 2 Transferases Each puts on and takes off groups AT (Adenylyltransferase - adenylylates GS) UT (Uridylyltransferase - uridylylates PII) One Regulatory Protein PII Two States AT-PII Adenylates AT-PII-UMP Deadenylylates

  11. Rules 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 deadenylylate

  12. AMP AT AT AT AT AT AMP GS PPi UTP PII PII PII PII PII PII UMP UMP UMP Uridylate removing Enzyme UR UR UT UT Active GS* ATP Less Active UMP = Useless = Uridylylates = Adenylylates (2 activities on same enzyme) = Deadenylylates = Deuridylylates

  13. Deadenylylates UTP PPi UR PII PII PII PII UMP UMP UR UMP UT UT Leads to adenylylation Activates GS ATP (-) glutamine (+) -Kg Inactivates GS Responsive to cell’s nitrogen requirements

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