Anaerobiospirillum succiniciproducens phosphoenolpyruvate carboxykinase. Mutagenesis at metal site 1

摘要

Anaerobiospirillum succiniciproducens phosphoenolpyruvate (PEP) carboxykinase catalyses the reversible metal-dependent formation of oxaloacetate (OAA) and ATP from PEP, ADP and CO_2. Mutations of PEP carboxykinase have been constructed where the residues His~(225) and Asp~(263), two residues of the enzyme's putative Mn~(2+) binding site, were altered. Kinetic studies of the His225Glu, and Asp263Glu PEP carboxykinases show 600- and 16 800-fold reductions in V_(max) relative to the wild-type enzyme, respectively, with minor alterations in K_m for Mn~(2+). Molecular modeling of wild-type and mutant enzymes suggests that the lower catalytic efficiency of the Asp263Glu enzyme could be explained by a movement of the lateral chain of Lys~(248), a critical catalytic residue, away from the reaction center. The effect on catalysis of introducing a negatively charged oxygen atom in place of N_(ε-2) at position 225 is discussed in terms of altered binding energy of the intermediate enolpyruvate.