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Activation of ribulose-1, 5-bisphosphate carboxylase by CO2 and Mg2+ is slow and reversible. At subsaturating concentrations of CO2 and Mg2+, positive effectors increase and negative effectors decrease the amount of active enzyme at equilibrium. Pre-equilibrium experiments indicated that both positive and negative effectors inhibit the rates of enzyme activation and deactivation. Greater than 99% inhibition of the activation and deactivation rates was observed at high effector concentrations, indicating that the binding and release of the activators CO2 and Mg2+ occur only with effector-free enzyme. The deactivation rate Ki values for the negative effector ribose 5-phosphate and the positive effectors inorganic phosphate, fructose 1,6-bisphosphate, and 6-phophogluconate were smaller than the corresponding activation rate Ki values by factors of 2, 6, 25, and 670, respectively. Thus, phosphorylated effectors impede deactivation more than activation. Equilibrium binding studies indicated that the active and inactive enzyme forms have similar affinities for the positive effectors inorganic phosphate (KD = 650 μM) and fructose bisphosphate (KD = 11 μM). The positive effector 3-phosphoglycerate was bound with greater affinity by the inactive enzyme (KD = 25 μM) than by the active enzyme (KD = 76 μM). Thus, preferential binding of positive effectors to the active enzyme form is not responsible for the enhancement of enzyme activation at equilibrium. The promotion of activation by positive effectors is mediated by altering the relative rates of activation and deactivation to favor active enzyme. Equilibrium binding studies indicated hat the inactive enzyme had a much greater affinity for ribose 5-phosphate (KD = 42 μM) than did the active enzyme (KD = 480 μM). Preferential binding of this negative effector to inactive enzyme exceeds its stabilizing effect on the active enzyme form and thus causes an overall reduction of activation at equilibrium.