Elucidation of the α-Ketoamide Inhibition Mechanism: Revealing the Critical Role of the Electrostatic Reorganization Effect of Asp17 in the Active Site of the 20S Proteasome

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.

Local Electric Fields Drives the Proton-Coupled Electron Transfer within Cytochrome P450 Reductase

“…Extensive experiments and theories have shown that externally applied electric fields or internal electric fields intrinsically present in certain catalysts can be used to effectively control chemical activity and selectivity. − Specifically, the significant role of electric fields has been extensively researched in both natural and synthetic enzymes. − Protonation of His180, adding a positive charge to the imidazole ring of its side chain, notably alters the electrostatic environment in which the PCET reaction takes place. We examined the electrostatic impact of the positively charged HIP180 by using Mulliken charges from QM/MM calculations to assess the interaction energy between His180’s charged side chain and the PCET reaction complex, including FADH – , FMN, Glu142, and the bridging water molecules.…”