On the mechanism of effector-mediated activation of ribulose bisphosphate carboxylase/oxygenase.

The slow, ordered, readily reversible binding of COZ and Mgl’ to ribulose bisphosphate carboxylase/oxygenase to form the active ternary enzyme-COz*Md+ complex has been studied at suboptimal COZ concentrations in the presence of various effectors. Sugar monophosphates, sugar bisphosphates, phosphate, and other anionic compounds either stimulated, inhibited, or had no effect on initial rates of carboxylation when preincubated with the enzyme, prior to assaying for catalytic activity. All of the positive effectors were rapidly equilibrating competitive inhibitors of the enzyme with respect to ribulose bisphosphates. Compounds that did not rapidly equilibrate were inhibitors. The mechanism of effector-mediated activation was studied by trapping the enzyme *COz-Mg’ complex with the tight binding inhibitor, carboxyarabinitol bisphosphate. Enzyme activation was dependent on effector concentration in the same manner as the formation of the enzyme COz* M g + carboxyarabinitol bisphosphate complex. It is concluded that positive effectors promote activation by stabilizing the active enzymeCOz-MgZ+ species rather than by any alternative method of activation. The model for effector-mediated activation requires only one effector site at which ribulose bisphosphate also binds. The model predicts that positive effectors should stimulate initial enzymatic rates only after preincubation with the enzyme under suboptimal COz and M 8 + concentrations, and only when the preincubated solution i s assayed after considerable dilution into solutions with high ribulose bisphosphate concentrations. Under steady state conditions of carboxylation, which are achieved only after several minutes of catalysis, effectors either have no influence or are inhibitory. Thus, effector-mediated initial enzyme activation is ultimately an order of addition and dilution.