¹H NMR T₁ relaxation rate study on substrate orientation of fluoromethylanilines in the active sites of microsomal and purified cytochromes P₄₅₀ 1A1 and 2B1

Abstract

The present study describes tH NMR T1 relaxation rate studies on fluoromethylanilines bound to the active sites of microsomal and purified cytoehromes P45o IAI and 2B1. From the data obtained, insights into the average orientation of the substrates with respect to the paramagnetic Fe 3÷ centre in the cytochromes P4so 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 ~r-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 Fe 3÷ 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 P45o 1A1 and 2B1 in a similar way. A time-averaged orientation of the substrate with the Fe 3+ above the aromatic ring, with the 7r-orbitals of the aromatic ring and those of the porphyrin rings in a parallel position, providing possibilities for energetically favourable ~t-~r 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 Fe 3÷ paramagnetie centre. The results obtained also indicate that possible differences in metabolite patterns resulting from conversion of the fluoromethylanilines by different cytochromes P450, especially P45o IAI and 2B1, are unlikely to be caused by a specific orientation of the substrate imposed by the substrate binding site of the enzyme.

Preferred Binding Orientations of Phenacetin in CYP1A1 and CYP1A2 Are Associated with Isoform-Selective Metabolism

“…In general, an increase in T 1 values for both substrates was observed after the reduction to the CO-complexed ferrous form of the enzyme (Tables 5 and 6) due to a higher diamagnetic contribution from the protein. This is in agreement with previous findings concerning T 1 relaxation times for CYP1A substrates, including earlier data for acetaminophen (Myers et al, 1994), as well as for fluoromethylanilines (Koerts et al, 1995) and caffeine (Regal and Nelson, 2000).…”

“…The measured distances for these aromatic protons were similar to each other, as well as to those of phenacetin and acetaminophen, thus suggesting that the ring orientations of the substrates examined are similar. In previous studies, similar observations for other planar CYP1A substrates, such as caffeine (Regal and Nelson, 2000) and fluoromethylanilines (Koerts et al, 1995), led to the proposal of plane-parallel binding orientations. However, this does not rule out other substrate binding orientations.…”

“…NMR studies with CYP1A isoforms have been successfully performed in the past and included binding of acetaminophen (Myers et al, 1994) and fluoromethylanilines (Koerts et al, 1995) to CYP1A1 and binding of caffeine to CYP1A2 (Regal and Nelson, 2000). In this study, we evaluated the binding interactions of substrates phenacetin and acetaminophen with CYP1A1 and CYP1A2.…”

Structural Features of Cytochromes P450 and Ligands that Affect Drug Metabolism as Revealed by X-Ray Crystallography and NMR

“…An NMR study was performed with structural isomers of methylfluoroaniline and these P450s to study regioselectivity and the mechanism of aniline metabolism [96]. The calculated distances from the NMR relaxation were within ~1 Å for all the structural isomers, leading the authors to hypothesize that the metabolism was not strongly influenced by the position of functional groups in the structures [96]. However, for aniline, which is N - and p -hydroxylated at opposite ends of the molecule, the molecule likely adopts multiple binding orientations within the P450 active site.…”

Modeling Kinetics of Subcellular Disposition of Chemicals

“…The relaxation induced by unpaired electrons provides information about the distance to the iron, 1397,1398 in addition to the identification of the interacting parts of the molecule. The proton−iron distances can be used as constraints for the docking of ligands to CYPs.…”