Abstract
The 20S proteasome is an attractive drug target for the development of anticancer agents because it plays an important role in cellular protein degradation. It has a threonine residue that can act as a nucleophile to attack inhibitors with an electrophilic warhead, forming a covalent adduct. Fundamental understanding of the reaction mechanism between covalent inhibitors and the proteasome may assist the design and refinement of compounds with the desired activity. In this study, we investigated the covalent inhibition mechanism of an α-keto phenylamide inhibitor of the proteasome. We calculated the noncovalent binding free energy using the PDLD/S-LRA/β method and the reaction free energy through the empirical valence bond method (EVB). Several possible reaction pathways were explored. Subsequently, we validated the calculated activation and reaction free energies of the most plausible pathways by performing kinetic experiments. Furthermore, the effects of different ionization states of Asp17 on the activation energy at each step were also discussed. The results revealed that the ionization states of Asp17 remarkably affect the activation energies and there is an electrostatic reorganization of Asp17 during the course of the reaction. Our results demonstrate the critical electrostatic effect of Asp17 in the active site of the 20S proteasome.