Characterization of the functional role of asp 141, asp 194, and asp464 residues in the Mn²⁺‐l‐malate binding of pigeon liver malic enzyme

Abstract

Pigeon liver malic enzyme was inactivated and cleaved at Asp141, Asp194, and Asp464 by the Cu 2ϩ -ascorbate system in acidic environment. Site-specific mutagenesis was performed at these putative metal-binding sites. Three point mutants, D141N, D194N, and D464N; three double mutants, D~141,194!N, D~194,464!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 Escherichia coli cells. All recombinant enzymes, except the triple mutant, were purified to apparent homogeneity by successive Q-Sepharose and adenosine-29,59-bisphosphate-agarose columns. The mutants showed similar apparent K m,NADP values to that of the WT. The K m,Mal value was increased in the D141N and D194N mutants. The K m,Mn value, on the other hand, was increased only in the D141N mutant by 14-fold, corresponding to ;1.6 kcal0mol for the Asp141-Mn 2ϩ binding energy. Substrate inhibition by l-malate was only observed in WT, D464N, and D~141,464!N. Initial velocity experiments 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 interactions between Asp141 and Asp194 in the metal binding that impel the D~141,194!N double mutant to an overall specificity constant @k cat 0~K d,Mn K m,Mal K m,NADP !# at least four orders of magnitude smaller than the WT value. This difference corresponds to an increase of 6.38 kcal0mol energy barrier for the catalytic efficiency. Mutation at Asp464, on the other hand, has partial additivity on the mutations at Asp141 and Asp194. The overall specificity constants for the double mutants D~194,464!N and D~141,464!N or the triple mutant D~141,194,464!N were decreased by only 10-to 100-fold compared to the WT. These results strongly suggest the involvement of Asp141 in the Mn 2ϩ -l-malate binding for the pigeon liver malic enzyme. The Asp194 and Asp464, which may be oxidized by nonspecific binding of Cu 2ϩ , are involved in the Mn 2ϩ -l-malate binding or catalysis indirectly by modulating the binding affinity of Asp141 with the Mn 2ϩ .

Multiple Cysteine Residues Are Implicated in Janus Kinase 2-Mediated Catalysis

“…Magnesium or manganese, in complex with ATP, is generally believed to be the physiologically relevant divalent cation bound to JAKs. While aspartate residues are known to coordinate manganese in some enzymes (42,43), the fact that cysteines can functionally substitute for aspartates in the coordination of Mn 2+ (44) raises the possibility that Cys866 and/or Cys917 might functionally coordinate the metallo-ATP substrate complex in JAK2. Two of the four cysteines, Cys1094 and Cys1105, affecting JAK2 activity are positionally conserved in 20 members of the Janus kinase family, including the insect orthologue HOP (Figure 7).…”