Probing the catalytic roles of Arg548 and Gln552 in the carboxyl-transferase domain of the Rhizobium etli pyruvate carboxylase by site-directed mutagenesis

摘要

The roles of Arg548 and Gln552 residues in the active site of the carboxyl transferase domain of Rhizobium etli pyruvate carboxylase were investigated using site-directed mutagenesis. Mutation of Arg548 to alanine or glutamine resulted in the destabilisation of the quaternary structure of the enzyme, suggesting that this residue has a structural role. The mutations R548K, Q552N and Q552A resulted in loss of the ability to catalyse pyruvate carboxylation, biotin-dependent decarboxylation of oxaloacetate and proton exchange between pyruvate and water. These mutants retained the ability to catalyse reactions that occur at the active site of the biotin carboxylase domain, i.e. bicarbonate-dependent ATP cleavage and ADP phosphorylation by carbamoyl phosphate. The effects of oxamate on the catalysis in the biotin carboxylase domain by the R548K and Q552N mutants were similar to those on the catalysis of reactions by the wild-type enzyme. However, the presence of oxamate had no effect on the reactions catalysed by the Q552A mutant. We propose that Arg548 and Gln552 facilitate the binding of pyruvate and subsequent proton transfer between pyruvate and biotin in the partial reaction catalysed in the active site of the carboxyl-transferase domain of Rhizobium etli pyruvate carboxylase.