title: Site-directed mutagenesis of catalytic residues in N(5)-carboxyaminoimidazole ribonucleotide synthetase.
abstract: N(5)-CAIR synthetase, an essential enzyme in microorganisms, converts 5-aminoimidazole ribonucleotide (AIR) and bicarbonate to N(5)-CAIR with the aid of ATP. Previous X-ray crystallographic analyses of Aspergillus clavatus N(5)-CAIR synthetase postulated that R271, H273, and K353 were important for bicarbonate binding and for catalysis. As reported here, site-directed mutagenesis of these residues revealed that R271 and H273 are, indeed, critical for bicarbonate binding and catalysis whereas all K353 mutations, even ones conservative in nature, are inactive. Studies on the R271K mutant protein revealed cooperative substrate inhibition for ATP with a Ki of 1.2 mM. Kinetic investigation of the H273A mutant protein indicated that it was cooperative with respect to AIR; however, this effect was not seen in either the wild-type or any of the other mutant proteins. Cooperative ATP-dependent inhibition of wild-type N(5)-CAIR synthetase was also detected with ATP displaying a Ki of 3.3 mM. Taken together, these results indicate that N(5)-CAIR synthetase operates maximally within a narrow concentration of ATP.
authors.affiliation: Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, Detroit, Michigan 48201, United States.
authors.forename: Mahender B
authors.initials: MB
authors.lastname: Dewal
authors.forename: Steven M
authors.initials: SM
authors.lastname: Firestine
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives*; D001639:Bicarbonates / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008025:Ligases / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D012265:Ribonucleotides / Q000096:biosynthesis*
chemical_list: D001639:Bicarbonates; D012265:Ribonucleotides; D000620:Aminoimidazole Carboxamide; D000255:Adenosine Triphosphate; D008025:Ligases; C031143:AICA ribonucleotide
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