Molecular orbital analysis of the catalytic process of serine proteinases: Effect of environment on protonation of the histidine aspartate diad of subtilisin

Abstract

The catalytic triad of serine proteinases is composed of serine, histidine and aspartate residues. During catalysis the imidazole group of histidine, as a general base, accepts the proton from the hydroxyl group of serine. In this work the equilibrium states of the proton between the protonated imidazole group and the aspartate ion, as well as the factors determining this equilibrium, are examined by various quantum chemical approximations. The protonated histidine-aspartate diad of subtilisin BPN' has been modelled by the imidazole-formic acid system. It is found that the CNDO/2 and ab initio STO-3G basis set calculations overestimate the stability of the neutral form where the proton is attached to the aspartate side chain. A more probable proton transfer energy is obtained if the total energy of the diad is partitioned into bond and interaction energy terms calculated with 4-31G and STO-3G basis sets, respectively. However, even this approximation is insufficiently accurate to establish unambiguously the position of the proton in the gas phase. Nevertheless, the effects cf the environment on proton transfer could be estimated with more certainty. It is found that each component of the environment, i.e. (1) amino-acid residues of subtilisin within 1 nm of the diad, (2) three water molecules and (3) a counter ion, stabilizes the imidazolium- aspartate ion pair of the diad so that the proton cannot be transferred from the imidazolium to the aspartate ion. These results clearly demostrate the crucial role of the environment in the reactivities of the catalytic group of enzymes.