PT - JOURNAL ARTICLE AU - J F Darbyshire AU - K R Iyer AU - J Grogan AU - K R Korzekwa AU - W F Trager TI - Substrate probe for the mechanism of aromatic hydroxylation catalyzed by cytochrome P450. DP - 1996 Sep 01 TA - Drug Metabolism and Disposition PG - 1038--1045 VI - 24 IP - 9 4099 - http://dmd.aspetjournals.org/content/24/9/1038.short 4100 - http://dmd.aspetjournals.org/content/24/9/1038.full SO - Drug Metab Dispos1996 Sep 01; 24 AB - The effect of branch pathways on the observed intramolecular isotope effect and deuterium retention associated with 6- and 7-hydroxylation of selectively monodeuterated (R)- and (S)-warfarin with cytochrome P450 (CYP) 2C9 and CYP1A2 were studied. cDNA-expressed CYP2C9 was incubated with enantiomerically pure (S)-7d1- and (S)-6d1-warfarin, and expressed CYP1A2 was incubated with enantiomerically pure (R)-7d1- and (R)-6d1-warfarin. A high degree of deuterium retention was observed in all metabolites, independent of the stereochemistry of the substrate or CYP isoform. No deuterium kinetic isotope effect was observed for the formation of 6-hydroxy- or 7-hydroxywarfarin in the case of the (S)-6d1-warfarin metabolism by CYP2C9, or for the formation of 6-hydroxy-, 7-hydroxy-, and 8-hydroxywarfarin in the case of the (R)-6d1-warfarin metabolism by CYP1A2. Deuterium isotope effects of 1.17 and 1.23 accompanied formation of 7-hydroxywarfarin from (S)-7d1-warfarin by CYP2C9 and from (R)-7d1-warfarin by CYP1A2, respectively. These observations are consistent with the addition-rearrangement pathway for aromatic hydroxylation, in which a triplet-like active oxygen species initially adds to the pi system, resulting in a tetrahedral intermediate. The intermediate subsequently rearranges to generate the phenol, the final product of the reaction.