Perturbation of reaction-intermediate partitioning by a site-directed mutant of ribulose-bisphosphate carboxylase/oxygenase.
To explore the roles of active-site Glu48 of ribulose-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum, the E48Q mutant has been characterized with respect to kinetics and product distribution. Although the kcat for carboxylase activity is only 0.6% of the wild-type value, the mutant retains full activity in catalyzing the conversion of the carboxylated reaction intermediate to 3-phosphoglycerate and retains 10% of the normal activity in catalyzing the enolization of ribulose bisphosphate. Thus, the mutant is preferentially impaired in the carboxylation step. Partitioning of the enediol(ate) intermediate during turnover of ribulose bisphosphate is perturbed dramatically in the case of the mutant protein. Whereas the wild-type enzyme displays a CO2/O2 specificity factor of 11, the corresponding parameter of the mutant is only 0.3, thereby signifying a shift of the relative reactivity of the enediol(ate) in favor of O2. The mutant protein is also unable to protect the enediol(ate) against misprotonation with consequential conversion of ribulose bisphosphate to xylulose bisphosphate. This side reaction, undetected with wild-type R. rubrum enzyme, proceeds as rapidly as carboxylation of D-ribulose 1,5-bisphosphate by the E48Q mutant. Formation of xylulose bisphosphate by the mutant does not appear to account for the decline in carboxylase activity that occurs during the course of an assay. These studies demonstrate the multiple functionalities of Glu48 in the facilitation of catalysis and in directing intermediate partitioning in the preferred direction.
Lee
E H
EH
Protein Engineering Program, Oak Ridge National Laboratory, Tennessee 37831-8077.
Harpel
M R
MR
Chen
Y R
YR
Hartman
F C
FC
D001665:Binding Sites; D002384:Catalysis; D002852:Chromatography, Ion Exchange; D003196:Computer Graphics; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D012247:Rhodospirillum rubrum / Q000201:enzymology; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity / Q000235:genetics
D012273:Ribulose-Bisphosphate Carboxylase