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Phenolic Hydroxyl Ionization in Two Subtilisins

From the ¹ From the Department of Biological Chemistry, UCLA School of Medicine, Los Angeles, California 90024

The ionization of the phenolic hydroxyl groups in subtilisins BPN' and Carlsberg has been studied by spectrophotometric titrations at 295 mµ between pH 7 and 13.7. Of the 10 tyrosine residues in subtilisin BPN', 5 are "exposed" and the remainder "buried." The ionization of the exposed phenolic hydroxyl groups is reversible with a pK_app of 9.74. In subtilisin Carlsberg, 6 residues appear to be exposed and the remaining 7 are buried. The exposed tyrosines ionize reversibly with a pK_app of 9.92. In 5 m guanidine hydrochloride, all of the tyrosine residues in both proteins titrate normally with a pK_app of 9.97. The buried tyrosine residues appear to be in two groups. The first contains 3 to 4 residues in BPN' or Carlsberg, respectively, with pK_app values of 11.25 to 11.6. In the second group there are 2 or 3 residues in BPN' or Carlsberg, respectively, all with pK_app values greater than 12.5.

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