title: Membrane anchor of cytochrome P450 reductase suppresses the uncoupling of cytochrome P450.
abstract: Cytochrome P450 reductase (CPR) is an important redox partner of microsomal CYPs. CPR is composed of a membrane anchor and a catalytic domain that contains FAD and flavin mononucleotide (FMN) as redox centers and mediates electron transfer to CYP. Although the CPR membrane anchor is believed to be requisite for interaction with CYP, its physiological role is still controversial. To clarify the role of the anchor, we constructed a mutant (Δ60-CPR) in which the N-terminal membrane anchor was truncated, and studied its effect on binding properties, electron transfer to CYP2C19, and drug metabolism. We found that Δ60-CPR could bind to and transfer electrons to CYP2C19 as efficiently as WT-CPR, even in the absence of lipid membrane. In accordance with this, Δ60-CPR could mediate metabolism of amitriptyline (AMT) and imipramine (IMP) in the absence of lipids, although activity was diminished. However, Δ60-CPR failed to metabolize omeprazole (OPZ) and lansoprazole (LPZ). To clarify the reason for this discrepancy in drug metabolism, we investigated the uncoupling reaction of the CYP catalytic cycle. By measuring the amount of H2O2 by-product, we found that shunt pathways were markedly activated in the presence of OPZ/LPZ in the Δ60-CPR mutant. Because H2O2 levels varied among the drugs, we conclude that the proton network in the distal pocket of CYP2C19 is perturbed differently by different drugs, and activated oxygen is degraded to become H2O2. Therefore, we propose a novel role for the membrane anchor as a suppressor of the uncoupling reaction in drug metabolism by CYP.
authors.affiliation: Graduate School of Pharmaceutical Sciences, Osaka University.
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Miyamoto
authors.forename: Taku
authors.initials: T
authors.lastname: Yamashita
authors.forename: Yuki
authors.initials: Y
authors.lastname: Yasuhara
authors.forename: Akinori
authors.initials: A
authors.lastname: Hayasaki
authors.forename: Yukari
authors.initials: Y
authors.lastname: Hosokawa
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Tsujino
authors.forename: Tadayuki
authors.initials: T
authors.lastname: Uno
mesh_terms: D000639:Amitriptyline / Q000737:chemistry / Q000378:metabolism; D055162:Biocatalysis; D065731:Cytochrome P-450 CYP2C19 / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000378:metabolism; D007099:Imipramine / Q000737:chemistry / Q000378:metabolism; D064747:Lansoprazole / Q000737:chemistry / Q000378:metabolism; D009154:Mutation; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009853:Omeprazole / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction
chemical_list: D064747:Lansoprazole; D000639:Amitriptyline; D006861:Hydrogen Peroxide; D065731:Cytochrome P-450 CYP2C19; D009251:NADPH-Ferrihemoprotein Reductase; D009853:Omeprazole; D007099:Imipramine