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

Ribulose diphosphate carboxylase was found to exist in two distinct kinetic forms in spinach leaf extracts. One form displayed an apparent K_m for CO₂ in excess of 200 μm and is likely to be the form purified and studied by many previous workers. However, if leaf extracts were prepared in the presence of Mg²⁺ and atmospheric levels of CO₂, the recently described high-affinity form was obtained. It had a K_m for CO₂ of about 20 μm, was quite stable even at 25 °C, and its properties were consistent with it being the form which operates in photosynthesis in vivo. Mg²⁺ was also able to convert the high-K_m (CO₂) form to the low-K_m (CO₂) form when it was added to an extract which had been prepared in its absence. Mg²⁺ 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 (CO₂) form is not intrinsically active and appears to have activity only by virtue of the low-K_m (CO₂) form produced by contact with Mg²⁺ and bicarbonate (or CO₂) during the course of the assay. Extracts prepared with ribose 5-phosphate in the absence of Mg₂₊ also showed low-K_m (CO₂) 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 (CO₂) form.Effects on the affinity of ribulose diphosphate carboxylase for CO₂ were paralleled by effects on the activity of the associated ribulose diphosphate oxygenase. Treatments which produced the low-K_m (CO₂) form of the carboxylase also resulted in high oxygenase activity, and it is possible that the apparently high-K_m (CO₂) form of the carboxylase has little, if any, oxygenase activity associated with it.The carboxylase and oxygenase activities of the low-K_m (CO₂) form showed broad and quite similar responses to pH variation, and the oxygenase had a K_m for O₂ of 0.22 mm.The stability of the low-K_m (CO₂) form in the presence of Mg²⁺ and bicarbonate was quite sufficient for it to be partially purified by Sepharose chromatography. The significance of the low-K_m (CO₂) form is discussed with respect to activation of photosynthesis by Mg²⁺.