title: Alteration of inhibitor selectivity by site-directed mutagenesis of Arg(294) in the ADP-glucose pyrophosphorylase from Anabaena PCC 7120.
abstract: Previous alanine scanning mutagenesis of ADP-glucose pyrophosphorylase from Anabaena PCC 7120 indicated that Arg(294) plays a role in inhibition by orthophosphate [J. Sheng, J. Preiss, Biochemistry 36 (1997) 13077]. In this study, analysis of several site-directed mutants in the presence of different metabolic effectors showed that the primary inhibitor for two of the mutant proteins, R294A and R294Q, was no longer orthophosphate but rather NADPH, which was a reversal in the pattern of inhibitor selectivity from the wild-type. Despite the differences in charge and size, analysis of the purified R294K, R294E, and R294Q mutant enzymes demonstrated similar decreases in orthophosphate affinity as the R294A mutant, while most of the other kinetic values were similar to those reported for the wild-type. All these results suggest that the positive charge of Arg(294) is not specifically involved in orthophosphate binding and that it is important in determining inhibitor selectivity.
authors.affiliation: Department of Biochemistry & Molecular Biology, Michigan State University, East Lansing, MI 48824, USA.
authors.forename: Jeremiah B
authors.initials: JB
authors.lastname: Frueauf
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Ballicora
authors.forename: Jack
authors.initials: J
authors.lastname: Preiss
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects; D019943:Amino Acid Substitution; D017033:Anabaena / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D051599:Glucose-1-Phosphate Adenylyltransferase; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000494:pharmacology; D009249:NADP / Q000494:pharmacology; D009713:Nucleotidyltransferases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics*; D010710:Phosphates / Q000494:pharmacology; D013329:Structure-Activity Relationship
chemical_list: D004791:Enzyme Inhibitors; D010710:Phosphates; D009243:NAD; D009249:NADP; D009713:Nucleotidyltransferases; D051599:Glucose-1-Phosphate Adenylyltransferase