title: Leucine 332 influences the CO2/O2 specificity factor of ribulose-1,5-bisphosphate carboxylase/oxygenase from Anacystis nidulans.
abstract: The role of Leu 332 in ribulose-1,5-bisphosphate carboxylase/oxygenase from the cyanobacterium Anacystis nidulans was investigated by site-directed mutagenesis. Substitutions of this residue with Met, Ile, Val, Thr, or Ala decreased the CO2/O2 specificity factor by as much as 67% and 96% for the Ile mutant in the presence of Mg2+ and Mn2+, respectively. For the Met, Ile, and Ala mutants in the presence of Mg2+, no loss of oxygenase activity was observed despite the loss of greater than 65% of the carboxylase activity relative to the wild-type enzyme. In the presence of Mn2+, carboxylase activities for mutant enzymes were reduced to approximately the same degree as was observed in the presence of Mg2+, although oxygenase activities were also reduced to similar extents as carboxylase activities. Only minor changes in Km(RuBP) were observed for all mutants in the presence of Mg2+ relative to the wild-type enzyme, indicating that Leu 332 does not function in RuBP binding. These results suggest that in the presence of Mg2+, Leu 332 contributes to the stabilization of the transition state for the carboxylase reaction, and demonstrate that it is possible to affect only one of the activities of this bifunctional enzyme.
authors.affiliation: Department of Biochemistry and Biophysics, Washington State University, Pullman 99164-4660.
authors.forename: G J
authors.initials: GJ
authors.lastname: Lee
authors.forename: K A
authors.initials: KA
authors.lastname: McDonald
authors.forename: B A
authors.initials: BA
authors.lastname: McFadden
mesh_terms: D002245:Carbon Dioxide / Q000378:metabolism*; D000458:Cyanobacteria / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D007930:Leucine / Q000235:genetics / Q000378:metabolism*; D008163:Luminescent Measurements; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D002245:Carbon Dioxide; D012273:Ribulose-Bisphosphate Carboxylase; D007930:Leucine; D010100:Oxygen