Metal ion binding and activation of Streptomyces griseus dinuclear aminopeptidase: cadmium(II) binding as a model

A detailed metal binding and activation of the dinuclear aminopeptidase fro m Streptomyces griseus (sAP) has been analyzed and modeled by means of meta l titration as well as kinetic and thermodynamic techniques using Cd2+ as a probe. Cd2+ binds to the two metal-binding sites in a sequential manner to produce a very active Cd2+-substituted derivative, particularly in the pre sence of Ca2+ (53% and 90%, respectively, relative to the activities of the native form in terms of k(cat)/K-m under the same conditions). The first s tepwise formation constant for the binding of metal to the dinuclear site ( to form M-sAP) was found to determine the metal-binding selectivity, regard less of the magnitude of the second stepwise formation constant (to form M, M-sAP from M-sAP). Interestingly, despite the seemingly very different bind ing profiles for different metal ions under different conditions, all of th em can be well described and fitted by the sequential binding model. In add ition, Ca2+ was found to significantly affect metal binding, inhibition, an d entropy of activation of this enzyme, and its role in sAP action is re-ev aluated.