title: Maize phosphoenolpyruvate carboxylase. Mutations at the putative binding site for glucose 6-phosphate caused desensitization and abolished responsiveness to regulatory phosphorylation.
abstract: Phosphoenolpyruvate carboxylases (PEPC, EC 4.1.1.31) from higher plants are regulated by both allosteric effects and reversible phosphorylation. Previous x-ray crystallographic analysis of Zea mays PEPC has revealed a binding site for sulfate ion, speculated to be the site for an allosteric activator, glucose 6-phosphate (Glc-6-P) (Matsumura, H., Xie, Y., Shirakata, S., Inoue, T., Yoshinaga, T., Ueno, Y., Izui, K., and Kai, Y. (2002) Structure (Lond.) 10, 1721-1730). Because kinetic experiments have also supported this notion, each of the four basic residues (Arg-183, -184, -231, and -372' on the adjacent subunit) located at or near the binding site was replaced by Gln, and the kinetic properties of recombinant mutant enzymes were investigated. Complete desensitization to Glc-6-P was observed for R183Q, R184Q, R183Q/R184Q (double mutant), and R372Q, as was a marked decrease in the sensitivity for R231Q. The heterotropic effect of Glc-6-P on an allosteric inhibitor, l-malate, was also abolished, but sensitivity to Gly, another allosteric activator of monocot PEPC, was essentially not affected, suggesting the distinctness of their binding sites. Considering the kinetic and structural data, Arg-183 and Arg-231 were suggested to be involved directly in the binding with phosphate group of Glc-6-P, and the residues Arg-184 and Arg-372 were thought to be involved in making up the site for Glc-6-P and/or in the transmission of an allosteric regulatory signal. Most unexpectedly, the mutant enzymes had almost lost responsiveness to regulatory phosphorylation at Ser-15. An apparent lack of kinetic competition between the phosphate groups of Glc-6-P and of phospho-Ser at 15 suggested the distinctness of their binding sites. The possible roles of these Arg residues are discussed.
authors.affiliation: Graduate School of Biostudies, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Akiko
authors.initials: A
authors.lastname: Takahashi-Terada
authors.forename: Masaaki
authors.initials: M
authors.lastname: Kotera
authors.forename: Kenta
authors.initials: K
authors.lastname: Ohshima
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Furumoto
authors.forename: Hiroyoshi
authors.initials: H
authors.lastname: Matsumura
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Kai
authors.forename: Katsura
authors.initials: K
authors.lastname: Izui
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D004789:Enzyme Activation; D019298:Glucose-6-Phosphate / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010730:Phosphoenolpyruvate Carboxylase / Q000737:chemistry*; D010766:Phosphorylation; D017433:Protein Structure, Secondary; D013431:Sulfates / Q000378:metabolism; D003313:Zea mays / Q000201:enzymology*
chemical_list: D013431:Sulfates; D019298:Glucose-6-Phosphate; D010730:Phosphoenolpyruvate Carboxylase