113Cd NMR of Cd(II)-substituted Zn(II) metalloenzymes.

Cadmium can replace zinc and magnesium in alkaline phosphatase from Escherichia coli, which permits the characterization of the catalytically important metal-binding sites by 113Cd NMR. At pH 6.5, in the absence of phosphate, two equivalents of cadmium are bound in identical sites (A), one in each monomer. Either raising the pH or phosphorylation of Cd2AP (AP is apoalkaline phosphatase) results in migration of Cd(II) from the site A in one monomer to the opposite monomer to occupy a second site (B) adjacent to the A site in the first monomer, a site stabilized by phosphorylation or high pH. At pH 6.5 in the presence of phosphate, the 113Cd NMR spectrum of Cd6AP consists of three narrow resonances from three pairs of fully occupied sites, A, B, and C. The resonances at 153 and 70 ppm represent two metal sites (A and B) 3.9 A apart at each active center and adjacent to the serine phosphorylated during turnover. At this pH the enzyme exists almost exclusively as the covalent phosphoseryl form E-P with a 31P resonance at approximately 9 ppm. As the pH is raised a 31P signal from the noncovalent E.P complex appears at approximately 13 ppm. This is reflected in the 113Cd spectrum by a split of both the A- and B-site resonances into pairs, a set at 137 and 65 ppm for E.P, and 153 and 70 ppm for the E-P species. 113Cd-O-31P coupling of 30 Hz on the 31P resonance of E.P shows the noncovalently bound phosphate to be coordinated to one but not both metal ions at each active site. The resonance of E-P is not coupled and thus the phosphoseryl residue appears to shift out of the coordination sphere of the active site metal ion.