Characterization of ribulose‐1,5‐bisphosphate carboxylase/oxygenase carrying ribulose 1,5‐bisphosphate at its regulatory sites and the mechanism of interaction of this form of the enzyme with ribulose‐1,5‐bisphosphate‐carboxylase/oxygenase activase

Ribulose‐1,5‐bisphosphate carboxylase/oxygenase [Rbu(1,5) P 2 CO] from plant sources shows a biphasic reaction course when assayed with more than 2 mM ribulose 1,5‐bisphosphate [Rbu(1,5) P 2 ]. In the burst, Rbu(1,5) P 2 CO has its substrate‐binding sites occupied with Rbu(1,5) P 2 for the initial few minutes, then both substrate‐binding and regulatory sites are occupied by Rbu(1,5) P 2 in the subsequent linear phase, at physiological concentrations of Rbu(1,5) P 2 [A. Yokota (1991) J. Biochem. (Tokyo) 110 , 246–252]. This study attempts the characterization of spinach Rbu(1,5) P 2 CO carrying Rbu(1,5) P 2 at the regulatory sites and the interaction of Rbu(1,5) P 2 CO activase with Rbu(1,5) P 2 CO purified with poly(ethylene glycol) 4000 without denaturation. Binding of Rbu(1,5) P 2 to the regulatory sites strongly influences the temperature dependence of the carboxylase activity of Rbu(1,5) P 2 CO. The activation energy of Rbu(1,5) P 2 CO with Rbu(1,5) P 2 at the regulatory sites was 40% larger than that without Rbu(1,5) P 2 over 30°C, although the binding did not affect the activation energy below this temperature. This caused the almost linear reaction course of the carboxylase reaction at 50°C. The optimum pH for the activity of Rbu(1,5) P 2 CO carrying Rbu(1,5) P 2 at the sites was 8.0–8.2, and increased by about pH 0.2 from that of Rbu(1,5) P 2 ‐CO without Rbu(1,5) P 2 . The ratio of the activity of the former form to that of the latter increased with increasing pH with an inflection point at pH 8.1. The increase in the ratio was accompanied by a decrease in the hysteric conformational change of Rbu(1,5) P 2 CO. The ATP‐hydrolyzing activity inherent to Rbu(1,5) P 2 CO activase was stimulated about twofold by 3–5 mM Rbu(1,5) P 2 . Rbu(1,5) P 2 CO in the inactive complex with Rbu(1,5) P 2 experienced hysteresis and bound Rbu(1,5) P 2 at the regulatory sites during activation in the presence of Rbu(1,5) P 2 CO activase. Evidence was obtained that Rbu(1,5) P 2 CO activase promoted the activation of Rbu(1,5) P 2 CO through binding to the large subunits of Rbu(1,5) P 2 CO.