Thermodynamic Fidelity of the Mammalian Cytochrome P450 2B4 Active Site in Binding Substrates and Inhibitors

Abstract

Structural plasticity of mammalian cytochromes P450 (CYP) has recently been explored in our laboratory and elsewhere to understand the ligand-binding promiscuity. CYP2B4 exhibits very different conformations and thermodynamic signatures in binding the small inhibitor 4-(4-chlorophenyl)imidazole (4-CPI) versus the large bifonazole. Using four key active-site mutants (F296A, T302A, I363A, and V367L) that are involved in binding one or both inhibitors, we dissected the thermodynamic basis for the ability of CYP2B4 to bind substrates and inhibitors of different sizes and chemistry. In all cases, 1:1 binding stoichiometry was observed. The inhibitors 4-CPI, 1-(4-chlorophenyl)imidazole, and 1-(2-(benzyloxy)ethyl)imidazole bind to the mutants with a free energy difference (ΔΔ G) of ∼ 0.5 to 1 kcal/mol compared with the wild type but with a large entropy–enthalpy compensation of up to 50 kcal/mol. The substrate testosterone binds to all four mutants with a ΔΔ G of ∼ 0.5 kcal/mol but with as much as 40 kcal/mol of entropy–enthalpy compensation. In contrast, benzphetamine binding to V367L and F296A is accompanied by a ΔΔ G of ∼ 1.5 and 3 kcal/mol, respectively. F296A, I363A, and V367L exhibit very different benzphetamine metabolite profiles, indicating the different substrate-binding orientations in the active site of each mutant. Overall, the findings indicate that malleability of the active site allows mammalian P450s to exhibit a high degree of thermodynamic fidelity in ligand binding.