Maize Leaf Phosphoenolpyruvate Carboxylase

Abstract

Maize (Zea mays L.) leaf phosphoenopyruvate (PEP) carboxylase activity at subsaturating levels of PEP was increased by the inclusion of glycerol (20%, v/v) in the assay medium. The extent of activation was dependent on H(+) concentration, being more marked at pH 7 (with activities 100% higher than in aqueous medium) than at pH 8 (20% activation). The determination of the substrate concentration necessary to achieve half-maximal enzyme activity (S(0.5)) (PEP) and maximal velocity (V) between pH 6.9 and 8.2 showed a uniform decrease in S(0.5) in the presence of glycerol over the entire pH range tested, and only a slight decrease in V at pH values near 8. Including NaCl (100 millimolar) in the glycerol containing assay medium resulted in additional activation, mainly due to an increase in V over the entire range of pH. Glucose-6-phosphate (5 millimolar) activated both the native and the glycerol-treated enzyme almost to the same extent, at pH 7 and 1 millimolar PEP. Inhibition by 5 millimolar malate at pH 7 and subsaturating PEP was considerably lower in the presence of glycerol than in an aqueous medium (8% against 25%, respectively). Size-exclusion high performance liquid chromatography in aqueous buffer revealed the existence of an equilibrium between the tetrameric and dimeric enzyme forms, which is displaced to the tetramer as the pH was increased from 7 to 8. In the presence of glycerol, only the 400 kilodalton tetrameric form was observed at pH 7 or 8. However, dissociation into dimers by NaCl could not be prevented by the polyol. We conclude that the control of the aggregation state by the metabolic status of the cell could be one regulatory mechanism of PEP carboxylase.