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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1975 Jan;72(1):394–397. doi: 10.1073/pnas.72.1.394

Cobalt(III), a probe of metal binding sites of Escherichia coli alkaline phosphatase.

R A Anderson, B L Vallee
PMCID: PMC432312  PMID: 164026

Abstract

To facilitate the study of individual metal binding sites of polymeric metalloproteins, conversion of exchange-labile Co(II) in E. coli alkaline phosphatase (EC 3.1.3.1) to exchange-inert Co(III) was examined. Oxidation of Co(II) alkaline phosphatase with hydrogen peroxide results in a single absorption maximum at 530 nm and loss both of the characteristic electron paramagnetic signal and of enzymatic activity. Zinc neither reactivates this enzyme nor displaces the oxidized cobalt atoms. Metal and amino-acid analyses demonstrate that oxidation alters neither cobalt binding nor amino-acid composition of the enzyme. Al data are consistent with the conclusion that hydrogen peroxide oxidizes Co(II) in alkaline phosphatase to Co(III). Polymeric metalloenzymes can contain different categories of metal atoms serving in catalysis, structure stabilization, and/or control and exerting their effects independently or interdependently. The in situ conversion of exchange-labile Co(II) to exchange-stable (Co(III) offers a method to selectively and differentially "freeze" cobalt atoms at their respective binding sites. The accompanying spectral changes and concomitant retardation in ligand exchange reactions may be used to differentiate between specific metal binding sites that serve different roles in polymeric metalloenzymes.

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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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