title: A functional analysis of the allosteric nucleotide monophosphate binding site of carbamoyl phosphate synthetase.
abstract: The catalytic activity of carbamoyl phosphate synthetase (CPS) from Escherichia coli is allosterically regulated by UMP, IMP, and ornithine. Thirteen amino acids within the domain that harbors the overlapping binding sites for IMP and UMP were mutated to alanine and characterized. The four residues that interact directly with the phosphate moiety of IMP in the X-ray crystal structure (K954, T974, T977, and K993) were shown to have the greatest impact on the dissociation constants for the binding of IMP and UMP and the associated allosteric effects on the kinetic constants of CPS. Of the four residues that interact with the ribose moiety of IMP (S948, N1015, T1017, and S1026), S1026 was shown to be more important for the binding of IMP than UMP. Five residues (V994, I1001, D1025, V1028, and I1029) were mutated in the region of the allosteric domain that surrounds the hypoxanthine ring of IMP. With the exception of V994A, these mutations had a modest influence on the binding and subsequent allosteric effects by UMP and IMP.
authors.affiliation: Department of Chemistry, Texas A&M University, College Station, Texas, 77842-3012
authors.forename: Olivier A
authors.initials: OA
authors.lastname: Pierrat
authors.forename: Frank M
authors.initials: FM
authors.lastname: Raushel
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000495:Allosteric Site; D001665:Binding Sites; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000737:chemistry* / Q000235:genetics; D002221:Carbamyl Phosphate / Q000378:metabolism; D018360:Crystallography, X-Ray; D005973:Glutamine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009711:Nucleotides / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary
chemical_list: D009711:Nucleotides; D005973:Glutamine; D002221:Carbamyl Phosphate; D000255:Adenosine Triphosphate; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)