pH dependence of the inhibition of chymotrypsin by a peptidyl trifluoromethyl ketone

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Abstract
The effects of pH on the kinetics of association and dissociation of chymotrypsin and the dipeptidyl trifluoromethyl ketone (TFK) N-acetyl-L-leucyl-L-phenylalanyltrifluoromethane (1) were examined through the pH range 4-9.5. The pH dependence of the association rate (kon) is similar to that of kcat/Km for ester and peptide substrates and is dependent on two pK''s at 7.0 and 8.9. We assign these pK''s to the active site His and to the amino group of the N-terminal isoleucine residue. Ki for the complex of 1 and chymotrypsin has a pH dependence very similar to that of kon, and we conclude that the same ionizable groups which determine the pH dependence of kon are involved. The dissociation constant of the enzyme-inhibitor complex (koff) shows no pH dependence between pH 4 and pH 9.5. The data indicate that the inhibitor reacts with a form of the enzyme in which His 57 is unprotonated, and the resulting complex contains no groups which ionize between pH 4 and pH 9.5. This is consistent with conclusions previously reached from NMR data (Liang and Abeles, 1987). These experiments led to the conclusion that 1 reacts with chymotrypsin to form a tetrahedral complex in which His 57 is protonated (pK > 9.5) and the OH group of serine 195 has added to the carbonyl group of 1 to form an ionized hemiketal (pK < 4.9). The pK of His 57 is increased by > 3 units over that in the free enzyme, and the pK of the hemiketal decreased by > 4 units compared to the pK in solution. We attribute the pK shifts to Coulombic interactions between the hemiketal oxyanion and the positively charged His, as well as to stabilization of the oxyanion by hydrogen-bond formation.