METAL-BINDING AND ACTIVE-SITE STRUCTURE OF DI-ZINC STREPTOMYCES-GRISEUS AMINOPEPTIDASE

Streptomyces griseus aminopeptidase has been characterized to have a d inuclear 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 h as 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 H-1 NMR spectrum with sharp hyperfi ne-shifted signals, again indicating the presence of a dinuclear activ e site. This H-1 NMR spectrum with sharp hyperfine-shifted features re presents a first of its kind for a di-Cu2+ center in metalloproteins.