Thermodynamic fidelity of the mammalian cytochrome P450 2B4 active site in binding substrates and inhibitors.

摘要: 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 thermodynamic signatures binding small inhibitor 4-(4-chlorophenyl)imidazole (4-CPI) versus large bifonazole. Using four key active-site mutants (F296A, T302A, I363A, V367L) that are involved one or both inhibitors, we dissected basis for ability bind substrates inhibitors sizes chemistry. In all cases, 1:1 stoichiometry was observed. The 4-CPI, 1-(4-chlorophenyl)imidazole, 1-(2-(benzyloxy)ethyl)imidazole with a free energy difference (ΔΔG) ∼ 0.5 1 kcal/mol compared wild type but entropy–enthalpy compensation up 50 kcal/mol. substrate testosterone binds ΔΔG ∼ 0.5 kcal/mol as much 40 kcal/mol compensation. contrast, benzphetamine V367L F296A is accompanied by ∼ 1.5 3 kcal/mol, respectively. F296A, exhibit metabolite profiles, indicating substrate-binding orientations active site each mutant. Overall, findings indicate malleability allows P450s high degree fidelity ligand binding.