The function of an enzyme depends on its dynamic structure, and the catalytic mechanism has long been an active focus of research. The principle for interpreting protein selectivity and fidelity stems from optimization of the active site upon protein–substrate complexation, i.e., a lock-and-key configuration, on which most protein–substrate molecule binding recognition, and hence drug discovery, relies. Yet another thought has been to incorporate the protein folding interior tunnels for stereo- and regio-selectivity along the protein–substrate or protein–ligand/inhibitor binding process. Free energy calculations provide valuable information for molecular recognition and protein–ligand binding dynamics and kinetics. In this study, we focused on the kinetics of cytochrome P450 proteins (CYP450s) and the protein interior tunnel structure–dynamics relationship in terms of the substrate binding and leaving mechanism. A case in point is given by the prostaglandin H2 (PGH2) homologous isomerase of prostacyclin synthase. To calculate the reactant and product traversing the tunnels to and from the heme site, the free energy paths and tunnel potentials of mean force are constructed from steered molecular dynamics simulations and adaptive basing force umbrella sampling simulations. We explore the binding tunnels and critical residue lining characteristics for the ligand traverse and the underlying mechanism of CYP450 activity. Our theoretical analysis provides insights into the decisive role of the substrate tunnel binding process of the CYP450 mechanism and may be useful in drug design and protein engineering contexts.

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