Strong hydrogen bonding in chymotrypsin and other serine proteases

The hydrogen bond linking His57‐Nδ1 and Asp102‐Oδ1 in chymotrypsin (Cht) at low pH and in transition state analogue complexes of Cht with peptide trifluoromethylketones (peptide‐TFKs) at pHs up to 12 has been assigned as a low barrier hydrogen bond (LBHB). The hydrogen bonds in these species of Cht display the physicochemical properties of LBHBs, as follows: 1) The proton NMR signals are far downfield, 18.1 ppm for Cht at low pH and 18.6–18.9 ppm for peptide‐TFK complexes. 2) The D/H fractionation factors are low, 0.3–0.4 for the peptide‐TFK complexes. 3) The deuterium and tritium isotope shifts (δ D –δ H , δ T –δ H ) are negative. 4) The enthalpies of activation for solvent exchange (ΔH ex ) are high, 10–19 kcal mol −1 . The LBHB is postulated to increase the base strength of His57 in the transition state for the nucleophilic addition of Ser195 to the peptide acyl group of a substrate. This property of His57 is displayed by His57 in the complexes of Cht with peptide‐TFKs, in which its p K a lies between 10.6 and 12 depending on the structure of the peptide. These values are optimal for an acid/base catalyst that both abstracts a proton from Ser195 in the formation of the tetrahedral intermediate and donates a proton to the leaving N ‐terminal amino group in the decomposition of the tetrahedral intermediate. Strong hydrogen bonds in simple molecules can be studied in both aqueous and nonaqueous solutions, and the conditions for their existence are discussed. Copyright © 2004 John Wiley & Sons, Ltd.