CYP2E1 competes with CYP2B4 for limited NADPH‐Cytochrome P450 Reductase

For efficient catalysis to occur, cytochromes P450 (CYPs or P450s) require an interaction with their physiological redox partner, cytochrome P450 reductase (CPR). However, CPR is in limited supply, as it exists in a 1:10 to 1:25 ratio to P450 in vivo . Furthermore, studies in both microsomal and reconstituted systems have shown that the presence of one P450 isoform can significantly influence the catalytic activity of another isoform. In this study, we assessed whether CYP2E1 could influence CYP2B4's catalytic properties under steady‐state turnover conditions. The results show that CYP2E1 can inhibit the CYP2B4‐mediated N ‐demethylation of benzphetamine (BNZ) with a K i of 0.4 μM. However, CYP2B4 was incapable of inhibiting CYP2E1‐mediated p‐nitrophenol hydroxylation. When these inhibition studies were performed with an artificial electron donor, tert‐butyl hydroperoxide, CYP2B4 was resistant to inhibition by CYP2E1. Determining the apparent K m of CYP2B4 for CPR in the presence of increasing concentrations of CYP2E1 revealed the dual competitive and non‐competitive nature of CYP2E1 inhibition since at ratios of 3:1 (CYP2E1:CYP2B4), CYP2E1 increased CYP2B4's K m by 13‐fold but had no effect on V max . Yet at higher ratios of CYP2E1:CYP2B4 (6:1), Km decreased to similar levels that were observed with CYP2B4 in the absence of CYP2E1; however, V max also decreased by 10‐fold. Additionally, CYP2E1 increased the K m of CYP2B4 for BNZ by 8‐fold, although the K m was recovered when saturating concentrations of CPR were used. These results demonstrate that CYP2E1 competes with CYP2B4 for limiting CPR and that this competition leads to significant changes in the K m and V max of CYP2B4 for CPR as well an elevated K m of CYP2B4 for BNZ. Further chemical cross‐linking studies are under way to determine if CYP2E1 interacts directly with CYP2B4 to prevent binding to CPR. (Supported in part by NIH grant CA16954).