Inactivation and conformational changes in methyl parathion hydrolase in 2,2,2-trifluoroethanol solutions: Inactivation kinetics and molecular dynamics simulation

Many improvements have been made in the understanding of functional and structural characteristics of proteins in a denaturant-based microenvironment. This study reports the chemical denaturation of methyl parathion hydrolase (MPH, EC 3.1.8.1) using 2,2,2-trifluoroethanol (TFE). MPH is an important enzyme that catalyzes the hydrolysis of organophosphorus agents. However, the regulation of MPH activity and structural changes during unfolding are not well studied, particularly for TFE unfolding. We investigated MPH unfolding with TFE for the first time. In this study, changes in enzymatic activity and unfolding of MPH at different TFE concentrations were investigated by enzyme activity measurements, intrinsic fluorescence and by 1-anilino-8-naphthalenesulfonate (ANS) fluorescence emission spectral scans. The results showed TFE inactivated MPH in a dose-dependent manner. A Lineweaver-Burk plot analysis revealed that the type of inhibition was reversible noncompetitive inhibition. Intrinsic fluorescence and ANS-binding fluorescence showed that TFE induced obvious tertiary structural changes in MPH by exposing hydrophobic groups. Furthermore, we conducted a docking simulation between MPH and TFE. The computer simulation successfully showed the binding structure and we estimated stability by calculating the binding energy (lowest binding energy: -3.18 kcal/mol). The results demonstrate that MPH can be inactivated by TFE, and provide new insights into the mechanism of TFE-induced unfolding of MPH and inhibition of ligand binding.