Mutational Analysis of C4-form Phosphoenolpyruvate Carboxylase: Identification of Arg Residues for Glucose 6-phosphate Binding and Their Participation in the Effect of Regulatory Phosphorylation

Abstract

Phosphoenolpyruvate carboxylase (PEPC) of higher plants is an allosteric enzyme being activated by glucose 6-phosphate (G6P) and inhibited by malate (MA) or aspartate. Monocot PEPC is also activated by glycine. Furthermore, the enzyme is activated by regulatory phosphorylation at Ser near the N-terminus. X-ray crystallographic analysis of maize C4-form PEPC pointed out the putative binding site for G6P. We show here that R183, R184, and R372 (of adjacent subunit), are involved in G6P activation by site-directed mutagenesis. Desensitization to glycine was also found with R372Q. The sensitivity to MA was decreased for R184Q, R183Q/R184Q, and R372Q. All of the mutant enzymes could be phosphorylated by a PEPC-specific protein kinase. However, changes in kinetic properties upon phosphorylation was lost or altered with R183Q, R184Q, R183Q/R184Q, and R372Q, indicating that these R residues participated not only in G6P binding but also somehow in molecular system of conformational change associated with the phosphorylation.