DISSOCIATION OF RIBULOSE-1,5-BISPHOSPHATE BOUND TO RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE OXYGENASE AND ITS ENHANCEMENT BY RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE OXYGENASE ACTIVASE-MEDIATED HYDROLYSIS OF ATP

Ribulose bisphosphate (RuBP), a substrate of ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco), is an inhibitor of Rubisco activation by carbamylation if bound to the inactive, noncarbamylated form of the enzyme. The effect of Rubisco activase on the dissociation kinetics of RuBP bound to this form of the enzyme was examined and characterized with the use of H-3-labeled RuBP and proteins purified from spinach (Spinacia oleracea L.) In the absence of Rubisco activase and in the presence of a large excess of unlabeled RuBP, the dissociation rate of bound [1-H-3]RuBP was much faster after a short (30 second) incubation than after an extended incubation (1 hour). After 1 hour of incubation, the dissociation rate constant (K(off)) of the bound RuBP was 4.8 x 10(-4) per second, equal to a half-time of about 35 minutes, whereas the rate after only 30 seconds was too fast to be accurately measured. This time-dependent change in the dissociation rate was reflected in the subsequent activation kinetics of Rubisco in the presence of RuBP, CO2, and Mg2+, and in both the absence or presence of Rubisco activase. However, the activation of Rubisco also proceeded relatively rapidly without Rubisco activase if the RuBP level decreased below the estimated catalytic site concentration. High pH (pH 8.5) and the presence of Mg2+ in the medium also enhanced the dissociation of the bound RuBP from Rubisco in the presence of RuBP. In the presence of Rubisco activase, Mg2+, ATP (but not the nonhydrolyzable analog, adenosine-5'-O-[3-thiotriphosphate]), excess RuBP, and an ATP-regenerating system, the dissociation of [1-H-3]RuBP from Rubisco was increased in proportion to the amount of Rubisco activase added. This result indicates that Rubisco activase-mediated hydrolysis of ATP is required for promotion of the enhanced dissociation of the bound RuBP from Rubisco. Furthermore, product analysis by ion-exchange chromatography demonstrated that the release of the bound RuBP, in an unchanged form, was considerably faster than the observed increase in Rubisco activity. Thus, RuBP dissociation was experimentally separated from activation and precedes the subsequent formation of active, carbamylated Rubisco during activation of Rubisco by Rubisco activase.