NMR investigations have been carried out of complexes between bovine chymotrypsin A, and a series of four peptidyl trifluoromethyl ketones, listed here in order of increasing affinity for chymotrypsin: N-Acetyl-L-Phe-CF3, N-Acetyl-Gly-L-Phe-CF3, N-Acetyl-L-Val-L-Phe-CF3, and N-AcetyI-L-Leu-L-Phe-CF3. The D/H fractionation factors (phi) for the hydrogen in the H-bond between His 57 and Asp 102 (His 57-H-delta 1) in these four complexes at 5 degrees C were in the range phi = 0.32-0.43, expected for a low-barrier hydrogen bond. For this series of complexes, measurements also were made of the chemical shifts of His 57-H-epsilon 1 (delta(2,2-dimethylsilapentane-5-sulfonic acid) 8.97-9.18), the exchange rate of the His 57-H-delta 1 proton with bulk water protons (284-12.4 s(-1)), and the activation enthalpies for this hydrogen exchange (14.7-19.4 kcal.mol(-1)). It was found that the previously noted correlations between the inhibition constants (K-i 170-1.2 mu M) and the chemical shifts Of His 57-H-delta 1 (delta(2,2-dimethylsilapentane-5-sulfonic acid) 18.61-18.95) for this series of peptidyl trifluoromethyl ketones with chymotrypsin [Lin, J., Cassidy, C. S. & Frey, P. A. (1998) Biochemistry 37, 11940-11948] could be extended to include the fractionation factors, hydrogen exchange rates, and hydrogen exchange activation enthalpies. The results support the proposal of low barrier hydrogen bond-facilitated general base catalysis in the addition of Ser 195 to the peptidyl carbonyl group of substrates in the mechanism of chymotrypsin-catalyzed peptide hydrolysis. Trends in the enthalpies for hydrogen exchange and the fractionation factors are consistent with a strong, double-minimum or single-web potential hydrogen bond in the strongest complexes. The lifetimes of His 57-H delta 1, which is solvent shielded in these complexes, track the strength of the hydrogen bond. Because these lifetimes are orders of magnitude shorter than those of the complexes themselves, the enzyme must have a pathway for hydrogen exchange at this site that is independent of dissociation of the complexes.
