![]() ![]() The thermodynamic signature obtained for structurally similar inhibitors suggests remarkable plasticity of CYP2B4. Steric restrictions hindered the perfect docking of only BEI to the closed conformation of the enzyme. Accessibility to acrylamide of the only tryptophan (Trp 121), which is located in helix C, was greatly decreased only in protein bound to 4-CPI. The largest difference in binding entropy (+5.9 versus -4.1 cal mol -1 K -1) was observed between 4-CPI and BEI, respectively, with a 2-fold difference in heat capacity changes (-604 versus -331 cal mol -1 K -1), which is inferred to result from the reduction of apolar surface area of the enzyme ensuing from a conformational change upon 4-CPI binding. Changes in enthalpy at 25 ☌ ranged from -6.5 to -8.8 kcal mol -1. Calorimetric titrations using monomeric enzyme yielded a 1:1 binding stoichiometry, with the associated K D values ranging from 0.3 to 4.8 μ m and following the same rank order as the IC 50 values. For example, usage of the Simplified Molecular Line Entry Specification (SMILES) format to represent lipid structures, while being very compact and accurate. Each of the inhibitors induced type II spectral changes, and IC 50 values for enzyme inhibition ranged from 0.1 to 2.4 μ m, following the order 1-BI < 4-CPI < 1-CPI < 4-PI < BEI < 1-PI. Here, we report for the first time a complete solution thermodynamic study using isothermal titration calorimetry supported by spectroscopic studies to elucidate the conformational flexibility of CYP2B4 in binding imidazole inhibitors with different ring chemistry and side chains: 4-CPI, 1-benzylimidazole (1-BI), 1-CPI, 4-phenylimidazole (4-PI), 1-(2-(benzyloxy)ethyl)imidazole (BEI), and 1-PI. Recent x-ray structures of cytochrome P450 2B4 (CYP2B4) reveal an open form that undergoes a large-scale structural transition to a closed form upon binding to 4-(4-chlorophenyl)imidazole (4-CPI). ![]()
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