The Enzymatic Carboxylation of Phosphoenolpyruvate

Abstract

Abstract Phosphoenolpyruvate carboxylase has been isolated from germinating peanut cotyledons and purified 2700-fold. The purified enzyme is approximately 80% pure as judged from sedimentation patterns, has a sedimentation coefficient (s20,w) of 13.9 S, and catalyzes the carboxylation of 50 µmoles of phosphoenolpyruvate per min per mg of protein (refractometrically determined) at 30°. Enzymatic carboxylation occurs optimally at pH 8.0 to 8.2. The Km values determined for HCO3-, phosphoenolpyruvate, and Mg2+ are 3.1 x 10-4, 5 to 6 x 10-4, and 3 to 4 x 10-4 m, respectively, at pH 7.9. Studies on the effect of pH on the Km values for phosphoenolpyruvate and Mg2+ were conducted. Evaluation of plots of -log Km (Mg2+ and phosphoenolpyruvate) against pH indicate that a dissociable group (or groups) of the carboxylase having a pK of 7.3, presumably an imidazole group, is involved in Mg2+ and phosphoenolpyruvate binding. Other evidence supporting this view is presented. During the enzymatic carboxylation of phosphoenolpyruvate, 18O from substrate HC18O3- is incorporated into the orthophosphate and oxalacetate reaction products in a ratio of 1:2. This indicates that the HCO3- (or CO32-) anion rather than CO2 is the active species in the carboxylation reaction. A mechanism is proposed which is consistent with this and other observations on the phosphoenolpyruvate carboxylase-catalyzed reaction.