The regulatory role of residues 226–232 in phosphoenolpyruvate carboxylase from maize
- 87 Downloads
The regulatory properties of maize phosphoenolpyruvate carboxylase were significantly altered by site-directed mutagenesis of residues 226 through 232. This conserved sequence element, RTDEIRR, is part of a surface loop at the dimer interface. Mutation of individual residues in this sequence caused various kinetic changes, including desensitization of the enzyme to key allosteric effectors or alteration of the K0.5 PEP for the substrate phosphoenolpyruvate. R231A, and especially R232Q, displayed decreased apparent affinity for the activator glucose-6-phosphate. Apparent affinity for the activator glycine was reduced in D228N and R232Q, while the maximum activation caused by glycine was greatly reduced in R226Q and E229A. R226Q and E229A also showed significantly lower sensitivity to the inhibitors malate and aspartate. E229A exhibited a low K0.5 PEP, while the K0.5 PEP of R232Q was significantly higher than that of wild type. Thus these seven residues are critical determinants of the enzyme’s kinetic responses to activators, inhibitors and substrate. The present results support an earlier suggestion that Arg 231 contributes to the binding site of the allosteric activator glucose-6-phosphate, and are consistent with other proposals that the substrate phosphoenolpyruvate allosterically activates the enzyme by binding at or near the glucose-6-phosphate site. The results also suggest that the glycine binding site may be contiguous with the glucose-6-phosphate binding site. Glu 229, which extends from this interface region through the interior of the protein and emerges near the aspartate binding site, may provide a physical link for propagating conformational changes between the allosteric activator and inhibitor binding regions.
Keywordsallosteric regulation C4 photosynthesis glucose-6-phosphate activation glycine activation malate inhibition phosphoenolpyruvate carboxylase site-directed mutagenesis
Crassulacean Acid Metabolism
This work was supported by NIH MBRS-RISE grant GM61331 and NIH Biomed-PREP grant GM 64104. The authors would like to thank Prof Vahe Bandarian for his assistance with this project.
- Gonzalez D, Iglesias A, Andreo C, (1984) On the regulation of phosphoenolpyruvate carboxylase activity from maize leaves by l-malate. Effect of pH J Plant Physiol 116: 425–434Google Scholar
- Sayegh J, Y. J, Sward L and Grover S (2002) Identification of arginine residues involved in activation of maize PEP carboxylase. Presented at Exp. Biology 2002, New Orleans 427.4Google Scholar
- Sutton F, Butler E, Smith T, (1986) Isolation of the structural gene encoding a mutant form of Escherichia coli phosphoenolpyruvate carboxylase deficient in regulation by fructose 1,6-bisphosphate. Identification of an amino acid substitution in the mutant J Biol Chem 261: 16078–16081PubMedGoogle Scholar
- Terada A, Kotera M, Tsumura K, Furumoto T, Matsumura H, Kai Y and Izui K (2001) Phosphoenolpyruvate carboxylase (PEPC): mutational analysis of a flexible loop and a putative binding site for an allosteric activator, glucose 6-phosphate (G6P). Presented at the 12th Interntl. Congr. Photosyn., Brisbane: S17-029Google Scholar
- Tovar-Mendez A, Rodriguez-Sotres R, Lopez-Valentin D, Munoz-Clares RA, (1998) Re-examination of the roles of PEP and Mg2+ in the reaction catalysed by the phosphorylated and non-phosphorylated forms of phosphoenolpyruvate carboxylase from leaves of Zea mays. Effects of the activators glucose 6-phosphate and glycine Biochem J 332: 633–632PubMedGoogle Scholar