Structure of chymotrypsin-trifluoromethyl ketone inhibitor complexes: comparison of slowly and rapidly equilibrating inhibitors

Abstract

The peptidyl trifluoromethyl ketones Ac-Phe-CF3 (1) and Ac-Leu-Phe-CF3 (2) are inhibitors of chymotrypsin. They differ in Ki (20 and 2 .mu.m, respectively) as well as in their kinetics of association with chymotrypsin in that 1 is rapidly equilibrating, with an association rate too fast to be observed by steady-state techniques, while 2 is "slow binding", as defined by Morrison and Walsh [Morrison, J. F., and Walsh, C. T. (1988) Adv. Enzymol. Relat. Areas Mol. Biol. 61, 202], with a second-order association rate constant of 750 M-1 s-1 at pH 7.0 [Imperiali, B., and Abeles, R. (1986) Biochemistry 25, 3760]. The crystallographic structures of the complexes of .gamma.-chymotrypsin with inhibitors 1 and 2 have been determined in order to establish whether structural or conformational differences can be found which account for different kinetic and thermodynamic properties of the two inhibitors. In both complexes, the active-site Ser 195 hydroxyl forms a covalent hemiketal adduct with the trifluoromethyl ketone moiety of the inhibitor. In both complexes, the trifluoromethyl group is partially immobilized, but differences are observed in the degree of interaction of fluorine atoms with the active-site His 57 imidazole ring, with amide nitrogen NH 193, and with other portions of the inhibitor molecule. The enhanced potency of Ac-Leu-Phe-CF3 relative to Ac-Phe-CF3 is accounted for by van der Waals interactions of the leucine side chain of the inhibitor with His 57 and Ile 99 side chains and by a hydrogen bond of the acetyl terminus with amide NH 216 of the enzyme. It is likely that the slower association rate of 2 with chymotrypsin as well as the lower Ki can be accounted for by the interaction of the P2 (Leu) substituent. No major difference in protein conformation between the two complexes were observed. Upon dissolution of crystalline chymotrypsin-Ac-Leu-Phe-CF3 into buffer, inhibitor is observed to dissociate from the complex at the same rate as observed for the enzyme-inhibitor complex which has been formed in solution, indicating that the complex studied crystallographically is probably identical with the complex which has been studied in solution [Imperiali, B., and Abeles, R. (1986) Biochemistry 25, 3760; Liang, T.-C., and Abeles, R. H. (1987) Biochemistry 26, 7603; Brady, K., Liang, T.-C., and Ables, R. H. (1989) Biochemistry 28, 9066; K. Brady and R. H. Abeles, submitted for publication].