1H NMR T 1 relaxation rate study on substrate orientation of fluoromethylanilines in the active sites of microsomal and purified cytochromes P450 1A1 and 2B1

FEBS Letters

Rietjens, Ivonne M.C.M.¹  Vervoort, Jacques¹  Koerts, Janneke¹  Boersma, Marelle G.¹ 

[1] Department of Biochemistry, Agricultural University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands

关键词: Cytochrome P450;    Fluoromethylaniline;    1H NMR;    T 1 relaxation measurement;   

DOI  :  10.1016/0014-5793(95)00672-V

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The present study describes ¹H NMR T ₁ relaxation rate studies on fluoromethylanilines bound to the active sites of microsomal and purified cytochromes P₄₅₀ 1A1 and 2B1. From the data obtained, insights into the average orientation of the substrates with respect to the paramagnetic Fe³⁺ centre in the cytochromes P₄₅₀ could be derived. Particular attention was paid to a possible extra relaxation pathway for methyl protons compared to the aromatic protons, due to the rotational motion of the CH₃ around the σ-C-CH₃ bond. However, this effect appeared to be minimal and to result in at most a few percent underestimation of the actual distance of the methyl protons to the Fe³⁺ centre. Furthermore, the data obtained demonstrate that all aromatic protons are at about the same average distance from the paramagnetic centre. The results also demonstrate that the fluoromethylanilines are bound to the active sites of cytochromes P₄₅₀ 1A1 and 2B1 in a similar way. A time-averaged orientation of the substrate with the Fe³⁺ above the aromatic ring, with the π-orbitals of the aromatic ring and those of the porphyrin rings in a parallel position, providing possibilities for energetically favourable π−π interaction defines the orientation which best fits the results of the present study. Possibilities for a flip-flop rotation around an axis in the plane of the aromatic ring can be included in this picture, as such rotations would still result in a similar average distance of all aromatic protons to the Fe³⁺ paramagnetic centre. The results obtained also indicate that possible differences in metabolite patterns resulting from conversion of the fluoromethylanilines by different cytochromes P₄₅₀, especially P₄₅₀ 1A1 and 2B1, are unlikely to be caused by a specific orientation of the substrate imposed by the substrate binding site of the enzyme.

【 授权许可】

Unknown