Effects of pH on Activity and Activation of Ribulose 1,5-Bisphosphate Carboxylase at Air Level CO(2).

The effects of pH on catalysis and activation characteristics of spinach ribulose 1,5-bisphosphate (RuBP) carboxylase were examined at air level of CO(2). Catalysis at limiting CO(2) was independent of pH over the range of pH 8.2 to 8.8 However, the kinetics of activation and the apparent equilibrium between the activated and inactivated forms of the enzyme were strongly dependent upon the pH and the presence or absence of the substrate RuBP. When incubated at air level of CO(2) at pH 8.2 in the absence of RuBP, the enzyme activation state was approximately 75% of that achieved with saturating CO(2) at that pH. The extent of activation increased with pH reaching 100% at pH values of 8.6 or higher. Adding RuBP to the activation medium after equilibrium activation state had been established decreased the apparent equilibrium activation level at pH values below 8.6. This effect was reversed at pH values above 8.6. Activation of inactive enzyme by CO(2) and Mg(2+) was inhibited dramatically at pH values below 8.6 and less so at pH values above 8.6. Studies showed that binding of RuBP to the inactive form of the enzyme was pH dependent with tighter binding occurring at lower pH values. It is suggested that the tight binding of RuBP to the inactive enzyme tends to decrease the equilibrium concentration of the activated form at pH values less than 8.6. These studies indicate that stromal pH could have a strong effect on the activation state of this enzyme in vivo, and possible feedback interactions which might adjust the apparent V(max) to match the rate of RuBP regeneration are discussed.