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-(3)H]RuBP is incubated with (14)CO(2) and O(2). Carboxylation rate is determined from (14)CO(2) incorporation and oxygenation rate is determined from [2-(3)H]glycolate-phosphate production. The assay was found to be suitable at all CO(2) and O(2) concentrations examined, which ranged from 0 to 300 micromolar CO(2) (20 millimolar NaHCO(3)) and 0 to 1.15 millimolar (100%) O(2). In combination with a polarographic assay, the stoichiometry of the RuBP oxygenase reaction was found to be RuBP-O(2)-glycolate phosphate-glycerate phosphate (1:1:1:1).The ratio of soybean (Merr.) RuBP carboxylase and oxygenase activities was measured at various CO(2) and O(2) 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 CO(2) and O(2) concentrations. The substrate specificity factor V(c)K(o)/V(o)K(c), which determines relative carboxylase and oxygenase reaction rates as a function of the CO(2) and O(2) concentrations, was found to be 73 in the presence of Mg(2+). Of the several compounds which have been reported to alter differentially the two reactions, we found that only Mn(2+) substitution for Mg(2+) was effective. Compared to Mg(2+), Mn(2+) reduced the K(m)(O(2)) from 690 to 36 micromolar O(2) and reduced the specificity factor to about 4. The K(m)(CO(2)) was about 20 micromolar CO(2) in the presence of both Mg(2+) and Mn(2+). 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 CO(2), with an activation equilibrium constant of about 1.3 millimolar NaHCO(3) (27 micromolar CO(2)) at pH 7.85 and 10 millimolar Mg(2+).