Identification of Amino Acid Substitutions that Confer a High Affinity for Sulfaphenazole Binding and a High Catalytic Efficiency for Warfarin Metabolism To P450 2C19

Abstract

Human cytochrome P450s 2C9 and 2C19 metabolize many important drugs including tolbutamide, phenytoin, and (S)-warfarin. Although they differ at only 43 of 490 amino acids, sulfaphenazole (SFZ) is a potent and selective inhibitor of P450 2C9 with an IC₅₀ and a spectrally determined binding constant, K_S, of K_S ∼ 4 μM) that was not seen when a shorter fragment of 2C9 was substituted (227−282). However, replacement of amino acids 283−338 resulted in extremely low holoenzyme expression levels in Escherichia coli, indicating protein instability. A single mutation, E241K, which homology modeling indicated would restore a favorable charge pair interaction between K241 in helix G and E288 in helix I, led to successful expression of this chimera that exhibited a K_S < 10 μM for SFZ. Systematic replacement of the remaining differing amino acids revealed that two amino acid substitutions in 2C19 (N286S, I289N) confer high-affinity SFZ binding (K_S < 5 μM). When combined with a third substitution, E241K, the resulting 2C19 triple mutant exhibited a high cataltyic efficiency for warfarin metabolism with the relaxed stereo- and regiospecificity of 2C19 and a lower K_M for (S)-warfarin metabolism (<10 μM) typical of 2C9.