The influence of pH on substrate form specificity of phosphoenolpyruvate carboxylase purified from Crassula argentea.
Purified phosphoenolpyruvate carboxylase from both the crassulacean acid metabolism plant Crassula argentea and the C4 plant Zea mays was shown by kinetic studies at saturating fixed-varying concentrations of free mg2+ to selectively use the metal-complexed form of phosphoenolpyruvate when assayed at pH 8.0. A similar response to added magnesium at high free phosphoenolpyruvate 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 phosphoenolpyruvate (including both free and metal-complexed forms) could be used by the enzyme from C.argentea while the C4 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 Km of this enzyme for phosphoenolpyruvate whatever the form considered and an increase in Vmax/Km. In contrast, a similar decrease of pH led to an increased Km of the C4 enzyme for phosphoenolpyruvate and a decrease of Vmax/Km. 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 phosphoenolpyruvate 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.
Meyer
C R
CR
Department of Biochemistry, University of California, Riverside 92521.
Rustin
P
P
Black
M K
MK
Wedding
R T
RT
D002262:Carboxy-Lyases / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010728:Phosphoenolpyruvate / Q000378:metabolism; D010730:Phosphoenolpyruvate Carboxylase / Q000302:isolation & purification*; D010944:Plants / Q000201:enzymology*; D013379:Substrate Specificity; D003313:Zea mays / Q000201:enzymology
D010728:Phosphoenolpyruvate; D002262:Carboxy-Lyases; D010730:Phosphoenolpyruvate Carboxylase; D008274:Magnesium