A Sensitive Assay Procedure for Simultaneous Determination of Ribulose-1,5-bisphosphate Carboxylase and Oxygenase Activities

Abstract

A sensitive assay procedure is described for the simultaneous determination of ribulose-1,5-bisphosphate (RuBP) carboxylase and oxygenase activities. In this assay, [1-³H]RuBP is incubated with ¹⁴CO₂ and O₂. Carboxylation rate is determined from ¹⁴CO₂ incorporation and oxygenation rate is determined from [2-³H]glycolate-phosphate production. The assay was found to be suitable at all CO₂ and O₂ concentrations examined, which ranged from 0 to 300 micromolar CO₂ (20 millimolar NaHCO₃) and 0 to 1.15 millimolar (100%) O₂. In combination with a polarographic assay, the stoichiometry of the RuBP oxygenase reaction was found to be RuBP-O₂-glycolate phosphate-glycerate phosphate (1:1:1:1). The ratio of soybean (Merr.) RuBP carboxylase and oxygenase activities was measured at various CO₂ and O₂ concentrations and in the presence of several compounds which have been reported to alter differentially the rate of the two reactions. The ratio of the two activities was a linear function of the ratio of the CO₂ and O₂ concentrations. The substrate specificity factor V_cK_o/V_oK_c, which determines relative carboxylase and oxygenase reaction rates as a function of the CO₂ and O₂ concentrations, was found to be 73 in the presence of Mg²⁺. Of the several compounds which have been reported to alter differentially the two reactions, we found that only Mn²⁺ substitution for Mg²⁺ was effective. Compared to Mg²⁺, Mn²⁺ reduced the K_m(O₂) from 690 to 36 micromolar O₂ and reduced the specificity factor to about 4. The K_m(CO₂) was about 20 micromolar CO₂ in the presence of both Mg²⁺ and Mn²⁺. Comparison of reaction rates in the presence of activated and inactivated enzyme allowed a direct determination that both carboxylase and oxygenase activities are similarly activated by CO₂, with an activation equilibrium constant of about 1.3 millimolar NaHCO₃ (27 micromolar CO₂) at pH 7.85 and 10 millimolar Mg²⁺.