CLONING, EXPRESSION, AND CHARACTERIZATION OF A ROOT-FORM PHOSPHOENOLPYRUVATE CARBOXYLASE FROM ZEA-MAYS - COMPARISON WITH THE C-4-FORM ENZYME

A full-length cDNA for maize root-form phosphoenolpyruvate carboxylase (PEPC) was isolated. In the coding region, the root-form PEPC showed 76 and 77% identity with the C-4- and C-3-form PEPCs of maize, respect ively, at the nucleotide level. At the amino acid level, the root-form was 81 and 85% identical to the C-4- and C-3-form PEPCs, respectively . The entire coding region was inserted into a pET32a expression vecto r so that it was expressed under the control of T7 promoter. The purif ied recombinant root-form PEPC had a V-max value of about 28 mu mol mi n(-1) (mg protein)(-1) at pH 8.0, The K-m values of root-form PEPC for PEP and Mg2+ were one-tenth or less of those of C-4-form PEPC when as sayed at either pH 7.3 or 8.0, while the value for HCO3- was about one -half of that of C-4- form PEPC at pH 8.0, Glucose 6-phosphate and gly cine had little effect on the root-form PEPC at pH 7.3; they caused tw o-fold activation of the C-4-form PEPC, The K-i (L-malate) values at p H 7.3 were 0.12 and 0.43 mM for the root- and C-4-form PEPCs, respecti vely. Comparison of hydropathy profiles among the maize PEPC isoforms suggested that several stretches of amino acid sequences may contribut e in some way to their characteristic kinetic properties. The root-for m PEPC was phosphorylated by both mammalian cAMP-dependent protein kin ase and maize leaf protein kinase, and the phosphorylated enzyme was l ess sensitive to L-malate.