Abstract
The two Zn(II) ions of native Escherichia coli alkaline phosphatase (EC 3.1.3.1) that are necessary for activity have been replaced by (63)Cu(II).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.
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