The Effect of Phosphorylated Metabolites and Divalent Cations on the Phosphatase and Carboxylase Activity of Maize Leaf Phosphoenolpyruvate Carboxylase

Abstract

Summary The modulation of maize leaf phosphoenolpyruvate (PEP) carboxylase activity by various phosphorylated metabolites and divalent cations was examined relative to phosphatase activity and synthesis of organic acids. The results are considered in relation to the two step reaction mechanism of the enzyme: Reaction of PEP and bicarbonate to form enol pyruvate and carboxyphosphate, and reaction of enol pyruvate with carboxyphosphate to form oxaloacetate and inorganic phosphate. At pH7, the highly purified enzyme exhibited phosphatase activities for glucose-6-phosphate and AMP of 12.5 and 5μmoles min -1 mg protein -1 , respectively. The formation of oxaloacetate by PEP carboxylase at pH7 and 3 mM PEP was stimulated by glucose-6-phosphate from 14 to 40μmoles min -1 mg protein -1 . Under similar conditions AMP also stimulated PEP carboxylase activity from 14 to 22μmoles min -1 mg protein -1 . In the case of glucose-6-phosphate, four binding sites were found per holoenzyme through the formation of a Schiff base, and a similar value was also obtained with PEP. The auxiliary phosphatase activity and stimulation of oxaloacetate synthesis are proposed to be due to maintenance of optimum levels of the carboxyphosphate intermediate in the reaction of PEP carboxylase. Among the divalent cations considered, Mg 2+ and Mn 2+ each caused a large increase in phosphatase activity with both glucose-6-phosphate and PEP as substrates. With PEP as substrate there was equivalent phosphatase and oxaloacetate synthesis. Zn 2+ strongly inhibited phosphatase activity. With Ca 2+ , Cd 2+ and Cu 2+ there was substantial phosphatase activity; however, they strongly inhibited carboxylation, i.e. the synthesis of oxaloacetate. A substantial amount of pyruvate was formed in the presence of these three divalent cations with PEP as substrate which is suggested to be derived from the catalytic enol-pyruvate intermediate.