Development of a method for the incorporation of substitution-inert metal ions into proteins. Site-specific modification of arsanilazotyrosine-248 carboxypeptidase A with cobalt(III)

Biochemistry
M S Urdea, J I Legg

Abstract

This investigation demonstrates the use of substitution-inert metal ions as site-specific amino acid modifying reagents. The approach involves the production of a chelating agent at the site of interest with the subsequent in situ oxidation of substitution-labile cobalt(II) to exchange-inert cobalt(III) with H2O2. We have produced the chelate complex ethylenediamine-N,N'-diacetato(arsanilazotyrosinato-248 carboxypeptidase A)cobalt(III) [CoIII(EDDA)(AA-CPA-Zn)]. Model CoIII(EDDA)(azophenolate) complexes have helped to define the reaction conditions necessary to produce the enzyme derivative and have proved invaluable in the spectral analysis of the cobalt(III)-enzyme complex. The modified enzyme contains one active-site zinc and one externally bound cobalt per enzyme monometer. Circular dichroism and visible spectra of the derivative and apoenzyme substantiate the site-specific nature of the incorporation. Concimitant with CoIIIEDDA incorporation, the enzyme loses its peptidase activity yet maintains with FeIIEDTA returns the original properties of the arsanilazotyrosine-248 enzyme.

References

Aug 1, 1986·Analytical Biochemistry·G J PielakJ I Legg

Citations

Jul 21, 1978·Biochimica Et Biophysica Acta·M GoreckiS Blumberg
Jan 1, 1976·Bioinorganic Chemistry·W I White, J I Legg
Jul 1, 1973·Proceedings of the National Academy of Sciences of the United States of America·J T Johansen, B L VALLEE
Oct 1, 1971·Proceedings of the National Academy of Sciences of the United States of America·J T Johansen, B L VALLEE
Oct 13, 1970·Biochemistry·G F Fairclough, B L VALLEE
Dec 1, 1964·Biochemistry·J E COLEMAN, B L VALLEE

Related Concepts

Carboxypeptidase A
EDDA
Hydrogen Peroxide
Apoenzymes
Complex (molecular entity)
Atoxyl
Chelating Agents
Carboxypeptidase A Activity
Plasma Protein Binding Capacity
Oxidation

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