Increased Phenacetin Oxidation upon the L382V Substitution in Cytochrome P450 1A2 is Associated with Altered Substrate Binding Orientation.

Leucine382 of cytochrome P450 1A2 (CYP1A2) plays an important role in binding and -dealkylation of phenacetin, with the L382V mutation increasing substrate oxidation (Huang and Szklarz, 2010, . 38:1039⁻1045). This was attributed to altered substrate binding orientation, but no direct experimental evidence had been available. Therefore, in the current studies, we employed nuclear magnetic resonance (NMR) longitudinal (T₁) relaxation measurements to investigate phenacetin binding orientations within the active site of CYP1A2 wild type (WT) and mutants. Paramagnetic relaxation time (T) for each proton of phenacetin was calculated from the T₁ value obtained from the enzymes in ferric and ferrous-CO state in the presence of phenacetin, and used to model the orientation of phenacetin in the active site. All aromatic protons of phenacetin were nearly equidistant from the heme iron (6.34⁻8.03 Å). In contrast, the distance between the proton of the ⁻OCH₂⁻ group, which is abstracted during phenacetin oxidation, and the heme iron, was much shorter in the L382V (5.93 Å) and L382V/N312L (5.96 Å) mutants compared to the N312L mutant (7.84 Å) and the wild type enzyme (6.55 Å), consistent with modeling results. These studies provide direct evidence for the molecular mechanism underlying increased oxidation of phenacetin upon the L382V mutation.