Anaerobiospirillum succiniciproducens phosphoenolpyruvate carboxykinase. Mutagenesis at metal site 1

Abstract

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.