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)

• Published in: Biochemistry (Easton) Vol. 18; no. 22; pp. 4984 - 4991
• Main Authors: Urdea, Mickey S, Legg, J. Ivan
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 01.10.1979
• Subjects: Amino Acids - analysis; Arsanilic Acid - analogs & derivatives; Arsanilic Acid - pharmacology; Carboxypeptidases - metabolism; Circular Dichroism; Cobalt - pharmacology; Hydrogen-Ion Concentration; Kinetics; Peptide Fragments - analysis; Protein Binding; Protein Conformation; Spectrophotometry; Tyrosine - analogs & derivatives; Tyrosine - pharmacology
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00589a028

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.