Metal-binding and active-site structure of di-zinc Streptomyces griseus aminopeptidase

Abstract

 Streptomyces griseus aminopeptidase has been characterized to have a dinuclear active site and to follow a dinuclear hydrolytic mechanism by means of activity assay, optical, and NMR spectroscopy. A sequential binding of Co²⁺ to the dinuclear sites in 20 mM Mes buffer at pH 6.1 has also been established. The results from these studies suggest that the two metal sites have a five-coordination sphere, with at least one coordinated His each. A di-Cu²⁺-substituted derivative of the enzyme has been prepared which exhibits a ¹H NMR spectrum with sharp hyperfine-shifted signals, again indicating the presence of a dinuclear active site. This ¹H NMR spectrum with sharp hyperfine-shifted features represents a first of its kind for a di-Cu²⁺ center in metalloproteins.