PERTURBATION OF REACTION-INTERMEDIATE PARTITIONING BY A SITE-DIRECTEDMUTANT OF RIBULOSE-BISPHOSPHATE CARBOXYLASE OXYGENASE

To explore the roles of active-site Glu48 of ribulose-bisphosphate car boxylase/oxygenase from Rhodospirillum rubrum, the E48Q mutant has bee n characterized with respect to kinetics and product distribution. Alt hough the k(cat) for carboxylase activity is only 0.6% of the wild-typ e value, the mutant retains full activity in catalyzing the conversion of the carboxylated reaction intermediate to 3-phosphoglycerate and r etains 10% of the normal activity in catalyzing the enolization of rib ulore bisphosphate. Thus, the mutant is preferentially impaired in the carboxylation step. Partitioning of the enediol(ate) intermediate dur ing turnover of ribulose bisphosphate is perturbed dramatically in the case of the mutant protein. Whereas the wild-type enzyme displays a C O2/O2 specificity factor of 11, the corresponding parameter of the mut ant 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 si de reaction, undetected with wild-type R. rubrum enzyme, proceeds as r apidly as carboxylation Of D-ribulose 1,5-bisphosphate by the E48Q mut ant. 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 functio nalities of Glu48 in the facilitation of catalysis and in directing in termediate partitioning in the preferred direction.