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 H 2 O 2 by-product, we found that shunt pathways were markedly activated in the presence of OPZ/LPZ in the Δ60-CPR mutant. Because H 2 O 2 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 H 2 O 2 . Therefore, we propose a novel role for the membrane anchor as a suppressor of the uncoupling reaction in drug metabolism by CYP.

The catalytic function of cytochrome P450 is entwined with its membrane-bound nature

“…29 found a correlation between the coupling efficiencies of CYP2J2 with the length of the TM domain of CPR: the more extensive the deletion of the TM helix, the lesser the coupling efficiencies. Similar results have been reported for CYP2C19 coupling with several substrates: full-length versus truncated CPR, as well as the presence or absence (or both) of lipids, was related to the overall catalytic efficiency 30 . Grinkova et al .…”

Effects of cytochrome P450 (CYP3A4 and CYP2C19) inhibition and induction on the exposure of selumetinib, a MEK1/2 inhibitor, in healthy subjects: results from two clinical trials

“…CYP3A4 is the pre-dominant isoform responsible for selumetinib oxidative metabolism with CYP1A2, CYP2C9, CYP2C19, CYP2E1, and CYP3A5 that is also involved to a smaller extent. CYP3A4, in particular, is involved in the metabolism of a wide range of compounds, including various drugs and toxins [4–6]. Exposure of selumetinib may be influenced by CYP inhibitors or inducers, in particular by inhibitors or inducers of CYP3A4 or CYP2C19, potentially affecting the safety profile or efficacy of selumetinib.…”

Effect of Drug Combination on Omeprazole Metabolism by Cytochrome P450 2C19 in <i>Helicobacter pylori</i> Eradication Therapy

“…CYP2C19 was expressed and purified as previously reported. 36) Recombinant human CYP reductase was prepared and its purity and activity were checked in accordance with a previously reported method. 37) Reduced nicotinamide adenine dinucleotide phosphate (NADPH) generating solution-containing 1 mM of β-nicotinamide-adenine dinucleotide phosphate (β-NADP + ), 2.5 mM of glucose-6-phosphate (G6P), and 2 U/mL of glucose-6-phosphate dehydrogenase (G6PDH) purchased from Oriental Yeast Co., Ltd. (Tokyo, Japan)-was prepared in 100 mM potassium phosphate buffer (KPi buffer, pH 7.4) with 2.5 mM MgCl 2 .…”

“…Assessment of Metabolic Activities The enzyme kinetic parameters (K m , V max , and CL int ) for OMP metabolism by CYP2C19 were measured by substrate depletion, as reported previously. 36) In addition, the effect of co-existing drugs on OMP metabolism by CYP2C19 was investigated by measuring the enzyme kinetic parameters in the presence of 629 µM CLR, 778 µM AMX, 872 µM TND, or 1250 µM MET. The concentrations were set to be 5-times higher than the evaluated K d values.…”