Metabolite control of Sorghum C4 phosphoenolpyruvate carboxylase catalytic activity and phosphorylation state

Kinetic analyses were performed on the nonphosphorylated and in vitro phosphorylated forms of recombinant Sorghum C-4 phosphoeno/pyruvate carboxylase (C-4 PEPC). The native enzyme was purified by immunoaffinity chromatography and its integrity demonstrated by Western blot analyses using anti N- and C-terminus antibodies. At suboptimal pH (7.1 to 7.3) and PEP concentration (2.5 mM), phosphorylation, positive metabolite effectors e.g., glucosc-6-phosphate, glycine and dihydroxyacetone-phosphate, or an increase in pH strongly activated the enzyme and lowered the inhibitory effect of L-malate. C-4 PEPC phosphorylation strengthened the effect of the positive effecters thereby decreasing further the enzyme's sensitivity to this inhibitor. L-malate also decreased the phosphorylation rate of C-4 PEPC, a process antagonized by positive metabolite effecters. This was shown both in vitro, in a reconstituted phosphorylation assay containing the catalytic subunit of a cAMP-dependent protein kinase or the Sorghum leaf PEPC-PK and in situ, during induction of C-4 PEPC phosphorylation in mesophyll cell protoplasts.