The role of cytochrome P450 2B6 and 2B4 substrate access channel residues predicted based on crystal structures of the amlodipine complexes

Abstract

Recent X-ray crystal structures of human cytochrome P450 2B6 and rabbit cytochrome P450 2B4 in complex with amlodipine showed two bound ligand molecules, one in the active site and one in the substrate access channel. Based on the X-ray crystal structures, we investigated the interactions of P450 2B4 and 2B6 with amlodipine using absorbance spectroscopy, and determined the steady-state kinetics of 7-ethoxy-4-(trifluoromethyl)coumarin and 7-benzyloxyresorufin oxidation by some access channel mutants to evaluate the functional role of these residues in substrate turnover. The results of absorbance titrations are consistent with a simple mechanism with two parallel binding events that result in the formation of the enzyme complex with two molecules of amlodipine. Using this model we were able to resolve two separate ligand-binding events, which are characterized by two distinct KD values in each enzyme. The access channel mutants R73K in P450 2B6 and R73K, V216W, L219W, and F220W in P450 2B4 showed a significant decrease in kcat/KM with the both substrates. Overall, the results suggest that P450 2B4 and 2B6 form an enzyme complex with two molecules of amlodipine in solution, and R73, V216, L219 and F220 in P450 2B4 may play an important role in substrate metabolism.

Mechanism of Drug–Drug Interactions Mediated by Human Cytochrome P450 CYP3A4 Monomer

“…The presence of two substrate molecules inside the substrate binding pocket can place one near the heme iron for metabolism, and the second in an unfavorable position to interact with the iron-oxo intermediate itself while in direct steric contact with the first. Examples of the latter type of binding can be seen in the X-ray structures of CYP3A4 with ketoconazole (61) and analogues of ritonavir (62, 63), and also in P450eryF with androstendione (70), CYP158A2 with flavioline (71), and in CYP2B4 and CYP2B6 structures (72, 73). In addition, dramatic acceleration of hydroxylation of small hydrocarbons and benzene by CYP102A1 has been demonstrated in the presence of a “decoy” effector molecule of perfluorodecanoic acid, which filled the active site cavity and stabilized binding of small hydrocarbon substrates near the active iron-oxygen intermediate (74, 75).…”

Interactions among Cytochromes P450 in Microsomal Membranes

“…The N-terminally truncated (⌬3-20) and C-terminally Histagged variant of CYP2E1 (38) was expressed in E. coli and purified following a procedure similar to that described for CYP2B6 (39) where the ion-exchange chromatography step was omitted. Modification of the proteins with thiol-reactive probes was performed as described previously (23).…”

Structure-Function Analysis of Mammalian CYP2B Enzymes Using 7-Substituted Coumarin Derivatives as Probes: Utility of Crystal Structures and Molecular Modeling in Understanding Xenobiotic Metabolism

“…In addition, site-directed mutagenesis was crucial to understand the structural and functional role of residues in the active site, access channel, and a recently observed peripheral pocket (Kobayashi et al, 1998;Hernandez et al, 2006;Jang et al, 2014Jang et al, , 2015. Our multifaceted approach has revealed how movement of secondary structure elements, such as the F-G helices and I helix, and reorientation of active-site residues, such as F206 and F297, allow CYP2B enzymes to bind compounds as small as a-pinene (molecular weight 5 136 g/mol) and as large as two molecules of amlodipine (molecular weight 5 409 g/mol) with high affinity.…”