Factors affecting interconversion between kinetic forms of ribulose diphosphate carboxylase-oxygenase from spinach

Abstract

Ribulose diphosphate carboxylase was found to exist in two distinct kinetic forms in spinach leaf extracts. One form displayed an apparent K m for CO 2 in excess of 200 μ m and is likely to be the form purified and studied by many previous workers. However, if leaf extracts were prepared in the presence of Mg 2+ and atmospheric levels of CO 2, the recently described high-affinity form was obtained. It had a K m for CO 2 of about 20 μ m, was quite stable even at 25 °C, and its properties were consistent with it being the form which operates in photosynthesis in vivo. Mg 2+ was also able to convert the high- K m (CO 2) form to the low- K m (CO 2) form when it was added to an extract which had been prepared in its absence. Mg 2+ was more effective in causing this conversion if bicarbonate was added as well. This activating effect of bicarbonate is a probable cause of previously reported apparent homotropic effects of bicarbonate on ribulose diphosphate carboxylase activity. It is possible that the apparently high- K m (CO 2) form is not intrinsically active and appears to have activity only by virtue of the low- K m (CO 2) form produced by contact with Mg 2+ and bicarbonate (or CO 2) during the course of the assay. Extracts prepared with ribose 5-phosphate in the absence of Mg 2+ also showed low- K m (CO 2) carboxylase activity initially, but the presence of this sugar phosphate was deleterious during storage at 25 °C, where it promoted conversion to the apparently high- K m (CO 2) form. Effects on the affinity of ribulose diphosphate carboxylase for CO 2 were paralleled by effects on the activity of the associated ribulose diphosphate oxygenase. Treatments which produced the low- K m (CO 2) form of the carboxylase also resulted in high oxygenase activity, and it is possible that the apparently high- K m (CO 2) form of the carboxylase has little, if any, oxygenase activity associated with it. The carboxylase and oxygenase activities of the low- K m (CO 2) form showed broad and quite similar responses to pH variation, and the oxygenase had a K m for O 2 of 0.22 m m. The stability of the low- K m (CO 2) form in the presence of Mg 2+ and bicarbonate was quite sufficient for it to be partially purified by Sepharose chromatography. The significance of the low- K m (CO 2) form is discussed with respect to activation of photosynthesis by Mg 2+.