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, KS, of <1 microM. P450 2C19 is not affected by SFZ at concentrations up to 100 microM. A panel of CYP2C9/2C19 chimeric proteins was constructed in order to identify the sequence differences that underlie this difference in SFZ binding. Replacement of amino acids 227-338 in 2C19 with the corresponding region of 2C9 resulted in high-affinity SFZ binding (KS approximately 4 microM) 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 KS < 10 microM 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 (KS < 5 microM). 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 KM for (S)-warfarin metabolism (<10 microM) typical of 2C9.