Dissection of the conduit for allosteric control of carbamoyl phosphate synthetase by ornithine.
Ornithine is an allosteric activator of carbamoyl phosphate synthetase (CPS) from Escherichia coli. Nine amino acids in the vicinity of the binding sites for ornithine and potassium were mutated to alanine, glutamine, or lysine. The residues E783, T1042, and T1043 were found to be primarily responsible for the binding of ornithine to CPS, while E783 and E892, located within the carbamate domain of the large subunit, were necessary for the transmission of the allosteric signals to the active site. In the K loop for the binding of the monovalent cation potassium, only E761 was crucial for the exhibition of the allosteric effects of ornithine, UMP, and IMP. The mutations H781K and S792K altered significantly the allosteric properties of ornithine, UMP, and IMP, possibly by modifying the conformation of the K-loop structure. Overall, these mutations affected the allosteric properties of ornithine and IMP more than those of UMP. The mutants S792K and D1041A altered the allosteric regulation by ornithine and IMP in a similar way, suggesting common features in the activation mechanism exhibited by these two effectors.
Pierrat
Olivier A
OA
Department of Chemistry, Texas A&M University, College Station, Texas 77842-3012, USA.
Javid-Majd
Farah
F
Raushel
Frank M
FM
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000409:Alanine / Q000737:chemistry; D000495:Allosteric Site; D001665:Binding Sites; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain; D002412:Cations; D004305:Dose-Response Relationship, Drug; D005973:Glutamine / Q000737:chemistry; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009952:Ornithine / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D011188:Potassium / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary
D002412:Cations; D005973:Glutamine; D000255:Adenosine Triphosphate; D009952:Ornithine; D000251:Adenosine Triphosphatases; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); D008239:Lysine; D000409:Alanine; D011188:Potassium