Characterization of the functional role of Asp141, Asp194, and Asp464 residues in the Mn2+-L-malate binding of pigeon liver malic enzyme

Pigeon liver malic enzyme was inactivated and cleaved at Asp141, Asp194, an d Asp464 by the Cu2+-ascorbate system in acidic environment. Site-specific mutagenesis was performed at these putative metal-binding sites. Three poin t mutants, D141N, D194N, and D464N; three double mutants, D(141,194)N, D(19 4,464)N, and D(141,464)N; and a triple mutant, D(141,194,464)N; as well as the wild-type malic enzyme (WT) were successfully cloned and expressed in E scherichia coli cells. All recombinant enzymes, except the triple mutant, w ere purified to apparent homogeneity by successive Q-Sepharose and adenosin e-2'5'-bisphosphate-agarose columns. The mutants showed similar apparent K- m,K-NADP values to that of the WT. The K-m,K-Mal value was increased in the D141N and D194N mutants. The K-m,K-Mn value, on the other hand, was increa sed only in the D141N mutant by 14-fold, corresponding to similar to 1.6 kc al/mol for the Asp 141-Mn2+ binding energy. Substrate inhibition by L-malat e was only observed in WT, D464N, and D(141,464)N. Initial velocity experim ents were performed to derive the various kinetic parameters. The possible interactions between Asp141, Asp194, and Asp464 were analyzed by the double -mutation cycles and triple-mutation box. There are synergistic weakening i nteractions between Asp141 and Asp194 in the metal binding that impel the D (141,194)N double mutant to an overall specificity constant [k(cat)/(Kd,MnK m,MalKm,NADP)] at least four orders of magnitude smaller than the WT value. This difference corresponds to an increase of 6.38 kcal/mol energy barrier for the catalytic efficiency. Mutation at Asp464, on the other hand, has p artial additivity on the mutations at Asp141 and Asp194. The overall specif icity constants for the double mutants D(194,464)N and D(141,464)N or the t riple mutant D(141,194,464)N were decreased by only 10- to 100-fold compare d to the WT. These results strongly suggest the involvement of Asp141 in th e Mn2+-L-malate binding for the pigeon liver malic enzyme. The Asp194 and A sp464, which may be oxidized by nonspecific binding of Cu2+ are involved in the Mn2+L-malate binding or catalysis indirectly by modulating the binding affinity of Asp141 with the Mn2+.