The influence of pH on substrate form specificity of phospho enolpyruvate carboxylase purified from Crassula argentea

Abstract

Purified phospho enolpyruvate carboxylase from both the crassulacean acid metabolism plant Crassula argentea and the C 4 plant Zea mays was shown by kinetic studies at saturating fixed-varying concentrations of free Mg 2+ to selectively use the metal-complexed form of phospho enolpyruvate when assayed at pH 8.0. A similar response to added magnesium at high free phospho enolpyruvate concentrations was obtained for both enzymes, consistent with the use of the complex as the substrate. Kinetic studies at pH 7.0 indicated that at this pH the total concentration of phospho enolpyruvate (including both free and metal-complexed forms) could be used by the enzyme from C. argentea while the C 4 enzyme still utilized the complex. The loss of specificity induced by the decrease in the pH of the assay medium was accompanied by a decrease in the K m of this enzyme for phospho enolpyruvate whatever the form considered and an increase in V max K m . In contrast, a similar decrease of pH led to an increased K m of the C 4 enzyme for phospho enolpyruvate and a decrease of V max K m . For the enzyme from C. argentea (previously shown to contain an essential arginine at the active site), protection of activity by the different forms of substrate against inactivation by the specific arginyl reagent 2,3-butanedione changes markedly with pH. At pH 8.1, the metal complex is the better protector while at pH 7.0 free phospho enolpyruvate gives the best protection consistent with the observed kinetic changes in substrate form utilization. The relationship between the enzyme affinity for substrate, substrate specificity, and the requirement for magnesium for substrate turnover is discussed.