Characterization of a low barrier hydrogen bond in the active site of chymotrypsin

Abstract

The possibility of strong hydrogen bonding in aqueous solutions is supported by spectroscopic and chemical properties of proton sponges and internally strained dicarboxylic acid monoanions in aqueous and aqueous/acetone solutions. A species of low barrier hydrogen bond (LBHB) in aqueous solutions is the downfield proton in transition state analogue tetrahedral addition complexes of chymotrypsin with peptide trifluromethylketones. The LBHB proton bridges His57 and Asp102 in the hemiketal adducts of Ser195 with the peptide trifluoromethylketones. The downfield proton resonances (18.6–18.9 ppm, the deterium isotope shift, the low deuterium fractionation factors (0.3–0.4), the close contact of His57 with Asp102 in crystal structures (≤2.6 Å), the high enthalpy of activation for exchange with solvent protons ( ΔH ex 014−19 kcal/ mol) and the high basicity of His57 (p K a 10.6–12) all support the assignment of an LBHB. In contrast to N-acetyl- l-leucyl- l-phenylalanine trifluoromethylketone, the comparable tetrahedral addition complex of the peptide aldehyde N-acetyl- l-leucyl- l-phenylalanal with Ser195 does not lead to an LBHB between His57 and Asp102 at pHs above 7. This is attributed to the high basicity of the hemiacetal adduct (p K a>13.5), so that the available proton in the active site resides on the hemiacetal hydroxyl group and not on His57.