Functional analysis of cytochrome P450 2B isoforms using ticlopidine as an active site probe

Abstract

A recent study of in vitro metabolism of ticlopidine by reconstituted human P450 2B6 and rabbit P450 2B4 indicated that both enzymes generated multiple and similar metabolites but in different ratios. In this study, we determined the metabolism of ticlopidine by additional cytochrome P450 2B enzymes, including rat P450 2B1 and dog P450 2B11. Based on the X‐ray crystal structure of the P450 2B4‐ticlopidine complex, two active site mutants, F206A and F297A were also created. All enzymes were expressed in E. coli as N‐terminal modified, and C‐terminal His‐tagged constructs termed 2B4dH. The separation and characterization of the different metabolites of ticlopidine were achieved using HPLC‐MS/MS. P450 2B1 and 2B11 produced ticlopidine N‐oxide as the major product, and the production of this metabolite by the P450 2B subfamily exhibited a rank order of 2B1 > 2B11 > 2B4 > 2B6. The pattern of the products observed for P450 2B1 and 2B11 was similar to that of P450 2B4. In contrast, the 2B4dH F206A and F297A mutants formed predominantly ticlopidine S‐oxide dimer previously seen with P450 2B6. The highest rate of the dimer formation was observed in F206A compared with other 2B isoforms including 2B4dH F297A. These results suggest that ticlopidine is a valuable probe for differentiating P450 2B subfamily members and for assessing the effect of site‐directed mutations.(Supported by NIH grant ES003619)