Insights in the peptide hydrolysis mechanism by thermolysin:: A theoretical QM/MM study

The hydrolysis by thermolysin of a Gly-Phe-Leu peptide, considered as a model substrate of the enkephalin family, has been studied with a mixed QM/MM method with the AM1/AMBER parameterization. This study is based on the mechanism proposed by Matthews in which the Glu-143 residue plays the role of a proton shuttle in the course of the reaction. The study focused on the description of every step of the process, reaction intermediates and transition states, and on the influence, both energetical and structural, of the whole protein on these stationary points. The overall mechanism appears to be quite realistic, but the study shows that some reaction steps that were assumed to be concerted should occur in two phases. Analysis of the role of the amino-acids surrounding the active site has shown their important participation in the fluctuations of the energy. In particular, the major role of His-231 on the overall mechanism has been confirmed. This study shows that modeling reaction mechanisms for enzymes is quite feasible and opens the way for computer experiments that may be helpful in devising and interpreting detailed experimental investigations.