LEUCINE-332 INFLUENCES THE CO2 O-2 SPECIFICITY FACTOR OF RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE OXYGENASE FROM ANACYSTIS-NIDULANS/

The role of Leu 332 in ribulose-1,5-bisphosphate carboxylase/oxygenase from the cyanobacterium Anacystis nidulans was investigated by site-d irected 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+, respecti vely. For the Met, Ile, and Ala mutants in the presence of Mg2+, no lo ss 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 r educed to approximately the same degree as was observed in the presenc e of Mg2+, although oxygenase activities were also reduced to similar extents as carboxylase activities. Only minor changes in K(m)(RuBP)) w ere observed for all mutants in the presence of Mg2+ relative to the w ild-type enzyme, indicating that Leu 332 does not function in RuBP bin ding. These results suggest that in the presence of Mg2+, Leu 332 cont ributes to the stabilization of the transition state for the carboxyla se reaction, and demonstrate that it is possible to affect only one of the activities of this bifunctional enzyme.