Selective Free Radical Reactions with Proteins and Enzymes: The Inactivation of Subtilisin Carlsberg and Subtilisin Novo

Abstract

Reactions of the selective inorganic radical anions (SCN)–2 and Br–2, with subtilisins Novo and Carlsberg lead to selective oxidation of the tryptophan, tyrosine, and histidine amino-acid residues. Inactivation studies show that most of the inactivation is caused by oxidation of one or more histidine residues. Oxidation of tryptophan residues has a minor effect upon activity but damage to tyrosine residues does not affect the activity of the two enzymes towards the small synthetic substrate N-acetyl-L-tyrosine ethyl ester. Rate constants for reactions with the radical anions and transient spectra, measured by pulse radiolysis, reflect the differences in amino-acid composition and in structure between the two enzymes.