Analysis of the Reaction Mechanism of Neuropsin Based on Dynamic Structure Sampling: MD×QM/MM Approach

The enzymatic reaction mechanism of the serine protease neuropsin (KLK8) was investigated using QM/MM calculations combined with MD simulations. The out-of-plane distortion of the scissile amide bond in the substrate-binding complex contributed to a reduction in the activation barrier. Nucleophilic attack by Ser195 Oγ initiated proton transfer among the catalytic triad and the formation of a tetrahedral intermediate. Free energy analysis indicated that this intermediate formation is the rate-determining step, emphasizing the importance of amide bond distortion and oxyanion hole stabilization for catalysis.