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).
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.
D000596:Amino Acids / Q000032:analysis; D001147:Arsanilic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D002268:Carboxypeptidases / Q000378:metabolism; D002942:Circular Dichroism; D003035:Cobalt / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D011487:Protein Conformation; D013053:Spectrophotometry; D014443:Tyrosine / Q000031:analogs & derivatives / Q000494:pharmacology; D010132:p-Azobenzenearsonate
D000596:Amino Acids; D010446:Peptide Fragments; C000229:tyrosine-4-azobenzenearsonate; D003035:Cobalt; D014443:Tyrosine; D010132:p-Azobenzenearsonate; D002268:Carboxypeptidases; D001147:Arsanilic Acid