| Summary: | Product regioselectivity as influenced by molecular recognition is a key aspect of enzyme catalysis. We applied large-scale two-dimensional (2D) umbrella sampling (USP) simulations to characterize acetaminophen (APAP) binding in the active sites of the family of Cytochrome P450 (CYP) enzymes as a case study to show the different regioselectivity exhibited by a single substrate in comparative enzymes. Our results successfully explain the experimentally observed product regioselectivity for all five human CYPs included in this study, demonstrating that binding events play an important role in determining regioselectivity. In CYP2C9 and CYP3A4, weak interactions in an overall large active site cavity result in a fairly small binding free energy difference between APAP reactive binding states, consistent with experimental results that show little preference for resulting metabolites. In contrast, in CYP1A2 and CYP2E1, APAP is strongly restrained by a compact binding pocket, leading to a preferred binding conformation. The calculated binding equilibrium of APAP within the compact active site of CYP2A6 is able to predict the experimentally documented product ratios and is also applied to explain APAP regioselectivity in CYP1A2 and CYP2C9. APAP regioselectivity seems to be related to the selectivity for one binding conformation over another binding conformation as dictated by the size and shape of the active site. Additionally, unlike docking and molecular dynamics (MD), our free energy calculations successfully reproduced a unique APAP pose in CYP3A4 that had been reported experimentally, suggesting this approach is well suited to find the realistic binding pose and the lowest-energy starting structure for studying the chemical reaction step in the future.
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