Acetylation of Tyrosine in Pepsin

In an earlier paper (1) the writer described the preparation and isolation of three crystalline acetyl derivatives of pepsin. They were, "100 per cent active" acetyl pepsin in which the 3 or 4 primary amino groups of pepsin had been acetylated with less than 15 per cent change in the specific activity; "60 per cent active" acetyl pepsin whichcontained 6-11 acetyl groups per molecule; and "10 per cent active" acetyl pepsin which had 20-30 acetyl groups per molecule of protein. Reversion of the 60 per cent active enzyme into the 100 per cent active was effected by treatment with normal sulfuric acid at 5°C. 1 It was pointed out in this earlier work that in all probability the primary amino groups belong to the lysine part of the protein molecule and that since acetylation of these groups failed to produce any appreciable effect on the activity of pepsin that the rSle played by the lysine in the enzymatic activity of the molecule is probably relatively small. Since the introduction of a few acetyl radicals into other groups of the protein molecule definitely diminished the specific activity, it was reasoned that these groups of the protein must be more closely related to the seat of the enzymatic activity. The present work was undertaken to determine the structural position of these few acetyl groups which have such a pronounced effect on the activity of pepsin. Though the evidence is somewhat indirect it nevertheless seems probable that those acetyl groups in the 60 per cent active acetyl pepsin which are responsible for the decrease in specific enzymatic activity are attached to the phenolic hydroxyl groups of some of the tyrosine components of the protein.