The deuterium isotope effect on the NMR signal of the low-barrier hydrogen bond in a transition-state analog complex of chymotrypsin.

The participation of a low-barrier hydrogen bond (LBHB) in the mechanism of action of chymotrypsin introduces a new role for Asp 102 and His 57 in catalysis [C. S. Cassidy, J. Lin, and P. A. Frey (1997) Biochemistry 36, 4576-4584]. It is postulated that the LBHB increases the basicity of His 57-N(epsilon2) in the transition state, thereby facilitating the abstraction of a proton from Ser 195, and stabilizes the tetrahedral intermediate in the acylation step. Evidence for this mechanism includes the downfield chemical shift of the proton bridging His 57 and Asp 102 in transition-state analog complexes and the low deuterium fractionation factors for this proton in the same complexes. We present additional spectroscopic evidence supporting the assignment of an LBHB between His 57 and Asp 102. The tetrahedral addition complex between Ser 195 of chymotrypsin and N-acetyl-l-leucyl-l-phenylalanyl trifluoromethylketone is regarded as a close structural analog of a tetrahedral intermediate. The deuterium NMR signal for the downfield deuteron bridging His 57 and Asp 102 in D(2)O has now been observed as a broad band centered at 17.8 +/- 0.5 ppm. The proton NMR signal in H(2)O is centered at 18.9 +/- 0.05 ppm. The two signals are clearly separated corresponding to a deuterium isotope effect of Delta[delta(H) - delta(D)] = 1.1 +/- 0.5 ppm. Deuterium isotope effects in this range are characteristic of LBHBs, and this observation provides further support for the assignment of the proton bridging His 57 and Asp 102 in transition-state analog complexes as an LBHB. Copyright 2000 Academic Press.