Spectroscopic studies of the exposure of tyrosine residues in proteins with special reference to the subtilisins.

The solvent perturbation behavior and acetylation characteristics of the tyrosine residues in the subtilisins have been examined. The availability of the phenolic side chains both to acetylation and solvent is high, in agreement with the proposed x-ray structure for subtilisin BPN’. Spectroscopic studies on N, 0-diacetyltyrosine, N-acetyltyrosine, and 3-nitrotyrosine in water-dioxane mixtures show that the spectra of these compounds are very sensitive to solvent composition. Consequently, when determination of the degree of acetylation, or nitration, is performed spectrophotometrically on native proteins, signxcant errors may occur due to dserences in the microenvironments of individual tyrosine side chains. Relevant data from the literature are discussed which indicate that phenolic groups in apolar locations undergo substitution preferentially, and that reactivity with N-acetylimidazole or tetranitromethane is an uncertain (and, in some cases, misleading) index of exposure of a phenolic side chain to the solvent. Circular dichroism studies on subtilisins Novo and Carlsberg indicate some differences in the arrangement of the aromatic residues in these two enzymes, and, possibly, small difIerences in the conformation of the polypeptide backbones as well.