KINETIC-ANALYSIS OF THE NONPHOSPHORYLATED, IN-VITRO PHOSPHORYLATED, AND PHOSPHORYLATION-SITE-MUTANT (ASP8) FORMS OF INTACT RECOMBINANT C-4 PHOSPHOENOLPYRUVATE CARBOXYLASE FROM SORGHUM

Steady-state kinetic analyses were performed on the non-phosphorylated, in vitro phosphorylated and phosphorylation-site mutant (Ser8-->Asp) forms of purified recombinant sorghum C-4, phosphoenolpyruvate (P-pyruvate) carboxylase (EC 4.1.1.31) containing an intact N-terminus. Significant differences in certain kinetic parameters were observed between these three enzyme forms when activity was assayed at a suboptimal but near-physiological pH (7.3), but not at optimal pH (8.0). Most notably, at pH 7.3 the apparent K-i for the negative allosteric effector L-malate was 0.17 mM, 1.2 mM and 0.45 mM while the apparent K-a for the positive allosteric effector glucose 6-phosphate (Glc6P) at 1 mM P-pyruvate was 1.3 mM, 0.28 mM and 0.45 mM for the dephosphorylated, phosphorylated and mutant forms of the enzyme, respectively. These and related kinetic analyses at pH 7.3 show that phosphorylation of C-4 P-pyruvate carboxylase near its N-terminus has a relatively minor effect on V and K-m (total P-pyruvate) but has a dramatic effect on the extent of activation by Glc6P, type of inhibition by L-malate and, most especially, K-a (Glc6P) and K-i (L-malate). Thus, regulatory phosphorylation profoundly influences the interactive allosteric properties of this cytosolic C-4-photosynthesis enzyme.