Study of the Groups Controlling the Activity and Conformation of Chymotrypsin at Alkaline pH: N‐Terminal Isoleucine and Tyrosines

It is shown that the change in activity and structure of acetyl‐δ‐chymotrypsin in the pH 7–11 range is not correlated with the ionizations of tyrosine residues. The pKs of the two titratable tyrosines of acetyl‐δ‐chymotrypsin are found to be 10.2 and 11.5, at 15° (9.6 and 10.8 after correction for electrostatic interactions), and those of the starting acetylated zymogen are 10.5 and 12.1 (10.0 and 11.5 after identical corrections). The plot of log rate of deacetylation of the titratable tyrosines versus pH is linear between pH 9 and pH 12. At a given pH there is a linear relation between the log deacetylation rate and the pKs of the residues. The pH rate profile of acylation of acetyl‐δ‐chymotrypsin by 4‐carboxy‐3‐nitrophenyl‐ N,N ‐diphenylcarbamate is bell‐shaped, with a maximum at pH 7.9 at 15°, in 0.14 M KCl. The right side of the curve shows a pK app of 8.9, whether the exposed titrable tyrosines of the enzyme are acetylated or not. A similar pK app (9.1) is found for the pH‐dependent change in optical rotation of the acetylated enzyme, when the exposed tyrosines are acetylated. After correction for electrostatic interactions, this apparent pK becomes 8.4 and can be assigned only to the α‐aminogroup of the N‐terminal isoleucine.