Understanding a Substrate’s Product Regioselectivity in a Family of Enzymes: A Case Study of Acetaminophen Binding in Cytochrome P450s

Abstract

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.

Linear Interaction Energy Based Prediction of Cytochrome P450 1A2 Binding Affinities with Reliability Estimation

“…Additionally, a recent computational study by Yang et al . [ 74 ] suggested Asn 312 , Asp 313 , and Phe 260 to be critical for binding of acetominophen to CYP 1A2.…”

Structural Features on the Substrate-Binding Surface of Fungal Lytic Polysaccharide Monooxygenases Determine Their Oxidative Regioselectivity

“…A study of cytochrome P450 (CYP) enzymes revealed that a large binding pocket results in less direct ligand‐protein interactions and a relatively fast interconversion between different binding states, resulting in low regioselectivity . The situation is even worse for most LPMOs as they have to rely on a binding surface instead of a pocket to accommodate the very large substrate.…”

Extracts from guava fruit protect renal tubular endothelial cells against acetaminophen‑induced cytotoxicity

“…Previous studies have demonstrated that cytochrome P450 enzymes can regulate cell proliferation (43,44). APAP interacts with the active sites of cytochrome P450 enzymes related to APAP-induced cytotoxicity (45,46). Many studies have shown that APAP-induced cytotoxicity requires bioactivation by cytochrome P450 enzymes (47,48), and APAP can induce the apoptotic death pathway by increasing cytochrome P450 activity (34).…”