Implication of an ionizing group in the control of conformation and activity of chymotrypsin.

1. Acetylated chymotrypsinogen was prepared by the reaction of crystallized chymotrypsinogen with acetic anhydride at pH 6.7 and 4O, and purification of the product. End group analysis and Van Slyke amino nitrogen determination indicated that all the free Eand or-amino groups of the chymotrypsinogen molecule had been blocked by acetylation. 2. Activation of acetylated chymotrypsinogen by trypsin, followed by chromatography, yielded acetylated d-chymotrypsin. Quantitative end group determination indicated that the molecule had a single amino group, the NH*-terminal a-amino group of an isoleucine residue. In the catalytic hydrolysis of N-acetyl-L-tryptophan amide, the steady state kinetic parameters were found to be the same, in the pH region 6 to 10, with acetylated d-chymotrypsin and with b-chymotrypsin. 3. The activation of acetylated chymotrypsinogen to acetylated &chymotrypsin was found to be accompanied by the appearance of a single new ionizing group, titratable in the pH region 5 to 10.5, with a pK(app) of 8.3. 4. The specific rotation of acetylated b-chymotrypsin was found to vary with pH in the pH region 6 to 10.5, the change in specific rotation following the ionization of a single group with pK(app) of 8.3. Above pH 10 the specific rotation of the zymogen and the enzyme were found to be the same at the three different wave lengths investigated. 5. Previous investigations in this and other laboratories of the pH dependence of the quotient of the steady state kinetic parameters, keat [K,(app)]-l-a function which yields the ionization constants of the catalytically important ionizing groups of the free enzyme -have indicated the importance of an ionizing group with pK(app) N 8.5 in all a-chymotrypsincatalyzed reactions studied, and in the 6or acetylated Schymotrypsin-catalyzed hydrolysis of N-acetyl-L-tryptophan amide. 6. An interpretation of these results that is consistent with all available experimental information is that the conversion