Catalytic Mechanism of Human T-Cell Leukemia Virus Type 1 Protease Investigated by Combined QM/MM Molecular Dynamics Simulations

Combined quantum mechanical and molecularmechanical(QM/MM) moleculardynamics simulations were performed to investigate the catalytic mechanismof human T-cell leukemia virus type 1 (HTLV-1) protease, a retroviralaspartic protease that is a potential therapeutic target for curingHTLV-1-associated diseases. To elucidate the proteolytic cleavagemechanism, we determined the two-dimensional free energy surfacesof the HTLV-1 protease-catalyzed reactions through various possiblepathways. The free energy simulations suggest that the catalytic reactionsof the HTLV-1 protease occur in the following sequential steps: (1)a proton is transferred from the lytic water to Asp32 ', followedby the nucleophilic addition of the resulting hydroxyl to the carbonylcarbon of the scissile bond, forming a tetrahedral oxyanion intermediate,and (2) a proton is transferred from Asp32 to the peptide nitrogenof the scissile bond, leading to the spontaneous breakage of the scissilebond. The rate-limiting step of this catalytic process is the protontransfer from Asp32 to the peptide nitrogen of the scissile bond,with a free energy of activation of 21.1 kcal/mol. This free energybarrier is close to the experimentally determined free energy of activation(16.3 kcal/mol) calculated from the measured catalytic rate constant(k (cat)). This mechanistic study providesdetailed dynamic and structural information that will facilitate thedesign of mechanism-based inhibitors for the treatment of HTLV-1-associateddiseases.