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. 1972 Apr;69(4):859–862. doi: 10.1073/pnas.69.4.859

Nitrogen Ligands at the Active Site of Alkaline Phosphatase

June S Taylor 1, Joseph E Coleman 1
PMCID: PMC426581  PMID: 4337243

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The two Zn(II) ions of native Escherichia coli alkaline phosphatase (EC 3.1.3.1) that are necessary for activity have been replaced by 63Cu(II).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 Az = 496 MHz, gz = gǁ = 2.27, and gx = gy = 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 Az 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.

Keywords: Zn, Cu, E. coli, electron spin resonance

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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