1H NMR T1 relaxation rate study on substrate orientation of fluoromethylanilines in the active sites of microsomal and purified cytochromes P450 1A1 and 2B1.
The present study describes 1H NMR T1 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 sigma-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 fluromethylanilines 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 pi-orbitals of the aromatic ring and those of the porphyrin rings in a parallel position, providing possibilities for energetically favourable pi-pi 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.
Koerts
J
J
Department of Biochemistry, Agricultural University, Wageningen, The Netherlands.
Rietjens
I M
IM
Boersma
M G
MG
Vervoort
J
J
D000814:Aniline Compounds / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001571:Benzoflavones / Q000494:pharmacology; D001665:Binding Sites; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D004790:Enzyme Induction; D017611:Fluorine Compounds / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008861:Microsomes / Q000201:enzymology*; D010634:Phenobarbital / Q000494:pharmacology; D011522:Protons; D051381:Rats; D013250:Steroid Hydroxylases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D019324:beta-Naphthoflavone
D000814:Aniline Compounds; D001571:Benzoflavones; D017611:Fluorine Compounds; D008024:Ligands; D011522:Protons; D019324:beta-Naphthoflavone; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C025619:steroid 16-beta-hydroxylase; D010634:Phenobarbital