Implication of an Ionizing Group in the Control of Conformation and Activity of Chymotrypsin

Abstract 1. Acetylated chymotrypsinogen was prepared by the reaction of crystallized chymotrypsinogen with acetic anhydride at pH 6.7 and 4°, and purification of the product. End group analysis and Van Slyke amino nitrogen determination indicated that all the free e- and α-amino groups of the chymotrypsinogen molecule had been blocked by acetylation. 2. Activation of acetylated chymotrypsinogen by trypsin, followed by chromatography, yielded acetylated δ-chymotrypsin. Quantitative end group determination indicated that the molecule had a single amino group, the NH2-terminal α-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 δ-chymotrypsin and with δ-chymotrypsin. 3. The activation of acetylated chymotrypsinogen to acetylated δ-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 δ-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, kcat Km(app)-1 —a function which yields the ionization constants of the catalytically importance ionizing groups of the free enzyme—have indicated the importance of an ionizing group with pK(app) ∼ 8.5 in all α-chymotrypsin-catalyzed reactions studied, and in the δ- or acetylated δ-chymotrypsin-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 of catalytically inactive chymotrypsinogen to active enzyme is accompanied by the appearance of a new ionizing group (a group that has a pK(app) of 8.3 in acetylated δ-chymotrypsin) which controls the conformation and thereby the activity of the enzyme. Molecules in which this ionizing group is unprotonated are in a catalytically inactive conformation, resembling that of chymotrypsinogen, and molecules in which this ionizing group is protonated are in a catalytically active conformation. A diisopropyl fluorophosphate (DFP)-induced protein isomerization involves only the enzyme molecules that are in the catalytically active conformation. This interpretation leads to a prediction that in the presence of DFP there will be an increase in pK(app) of the ionizing group. Such an increase in pK(app) was previously observed, and accounts for the proton uptake and the change in specific rotation of the enzyme observed in its reaction with DFP. 7. All evidence obtained so far suggests that the group with pK(app) ∼8.5 is an NH2-terminal isoleucyl α-amino group.