Purification and properties of phosphoenolpyruvate carboxylase from the germinating peanut cotyledon

Abstract

Phosphoenolpyruvate carboxylase has been partially (50-fold) from extracts of germinating peanut cotyledons. The enzyme catalyzes the irreversible Mg 2+-dependent carboxylation of phosphoenolpyruvate to form oxaloacetate. Purified preparations of the carboxylase are completely inactive in the absence of added sulfhydryl compounds, but are readily reactivated by glutathione addition. Carboxylase, active in the presence of small amounts of glutathione, is reversibly inhibited by p-hydroxymercuribenzoate. Co 2+ and Mn 2+ are 22% and 10% as effective, respectively, as equimolar amounts of Mg 2+ for the carboxylation reaction. In the absence of phosphoenolpyruvate, high levels of purified carboxylase failed to catalyze significant incorporation of [ 14C]bicarbonate into oxaloacetate in the presence of oxaloacetate, orthophosphate, Mg 2+ and glutathione. When the enzymic carboxylation of phosphoenolpyruvate was conducted in the presence of [ 14C]pyruvate, incorporation of 14C-activity into oxaloacetate did not occur. The catalytic action of the carboxylase is not inhibited by avidin.