Fractionation factors and activation energies for exchange of the low barrier hydrogen bonding proton in peptidyl trifluoromethyl ketone complexes ofchymotrypsin

NMR investigations have been carried out of complexes between bovine chymot rypsin A, and a series of four peptidyl trifluoromethyl ketones, listed her e in order of increasing affinity for chymotrypsin: N-Acetyl-L-Phe-CF3, N-A cetyl-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 betwee n His 57 and Asp 102 (His 57-H-delta 1) in these four complexes at 5 degree s 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 chem ical 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 bu lk water protons (284-12.4 s(-1)), and the activation enthalpies for this h ydrogen 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-s ulfonic acid) 18.61-18.95) for this series of peptidyl trifluoromethyl keto nes with chymotrypsin [Lin, J., Cassidy, C. S. & Frey, P. A. (1998) Biochem istry 37, 11940-11948] could be extended to include the fractionation facto rs, hydrogen exchange rates, and hydrogen exchange activation enthalpies. T he results support the proposal of low barrier hydrogen bond-facilitated ge neral base catalysis in the addition of Ser 195 to the peptidyl carbonyl gr oup of substrates in the mechanism of chymotrypsin-catalyzed peptide hydrol ysis. Trends in the enthalpies for hydrogen exchange and the fractionation factors are consistent with a strong, double-minimum or single-web potentia l 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 tha n those of the complexes themselves, the enzyme must have a pathway for hyd rogen exchange at this site that is independent of dissociation of the comp lexes.