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Leucine 332 influences the CO 2 /O 2 specificity factor of ribulose‐1, 5‐bisphosphate carboxylase/oxygenase from Anacystis nidulans
Author(s) -
Lee Garrett J.,
McDonald Kelly A.,
McFadden Bruce A.
Publication year - 1993
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560020709
Subject(s) - oxygenase , ribulose 1,5 bisphosphate , pyruvate carboxylase , chemistry , biochemistry , rubisco , cyanobacteria , ribulose , enzyme , biology , bacteria , genetics
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 CO 2 /O 2 specificity factor by as much as 67% and 96% for the Ile mutant in the presence of Mg 2+ and Mn 2+ , respectively. For the Met, Ile, and Ala mutants in the presence of Mg 2+ , 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 Mn 2+ , carboxylase activities for mutant enzymes were reduced to approximately the same degree as was observed in the presence of Mg 2+ , although oxygenase activities were also reduced to similar extents as carboxylase activities. Only minor changes in K m (RuBp) were observed for all mutants in the presence of Mg 2+ relative to the wild‐type enzyme, indicating that Leu 332 does not function in RuBP binding. These results suggest that in the presence of Mg 2+ , 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.