Nitrogen ligands at the active site of alkaline phosphatase.

The two Zn(II) ions of native Escherichia coli alkaline phosphatase (EC 3.1.3.1) that are necessary for activity have been replaced by (63)Cu(II). Titration of apoenzyme with up to 2 eq of Cu(II) gives a homogeneous species with an electron spin resonance typical for Cu(II) in an axially symmetric environment, with A(z) = 496 MHz, g(z) = g = 2.27, and g(x) = g(y) = 2.05. At least seven nitrogen hyperfine lines, spaced 11 G apart, are clearly resolved on the M = +[unk] Cu(II) hyperfine peak in the parallel region. When more than 2 eq of Cu(II) are added, the electron spin resonance spectrum shows at least two types of Cu(II) binding sites; the additional site, or sites, are characterized by lower g and higher A(z) values. When Cu(II) is added to native Zn(II) alkaline phosphatase or to apoenzyme incubated with 2 eq of Zn(II), the electron spin resonance spectrum shows little or no trace of the species with higher g values and nitrogen splitting. These results indicate that the species with higher g represents copper bound at the site normally occupied by the 2 Zn (II) ions necessary for enzyme activity, and that the metal ion at this site has at least 3 equivalent nitrogen ligands, probably histidyl side chains.