Global and Kinetic Profiles of Substrate Diffusion in Candida antarctica Lipase B: Molecular Dynamics with the Markov-State Model

[Image: see text] Profiling substrate diffusion pathways with kinetic information, which accounts for the dynamic nature of enzyme–substrate interaction, can enable molecular reengineering of enzymes and process optimization of enzymatic catalysis. Candida antarctica lipase B (CALB) is extensively used for producing various chemicals because of its rich catalytic mechanisms, broad substrate spectrum, thermal stability, and tolerance to organic solvents. In this study, an all-atom molecular dynamics (MD) combined with Markov-state models (MSMs) implemented in pyEMMA was proposed to simulate diffusion pathways of 4-nitrophenyl ester (4NPE), a commonly used substrate, from the surface into the active site of CALB. Six important metastable conformations of CALB were identified in the diffusion process, including a closed state. An induced-fit mechanism incorporating multiple pathways with molecular information was proposed, which might find unprecedented applications for the rational design of lipase for green catalysis.