IDENTIFICATION OF AMINO-ACID SUBSTITUTIONS THAT CONFER A HIGH-AFFINITY FOR SULFAPHENAZOLE BINDING AND A HIGH CATALYTIC EFFICIENCY FOR WARFARIN METABOLISM TO P450 2C19

Human cytochrome P450s 2C9 and 2C19 metabolize many important drugs in cluding tolbutamide, phenytoin, and (S)-warfarin. Although they differ at only 43 of 490 amino acids, sulfaphenazole (SFZ) is a potent and s elective inhibitor of P450 2C9 with an IC50 and a spectrally determine d binding constant, K-S, of <1 mu M. P450 2C19 is not affected by SFZ at concentrations up to 100 mu M. A panel of CYP2C9/2C19 chimeric prot eins was constructed in order to identify the sequence differences tha t 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 (K-S similar to 4 mu M) that was not seen when a shorter fragment of 2C9 was substituted (227-282). However, replacemen t of amino acids 283-338 resulted in extremely low holoenzyme expressi on levels in Escherichia coli, indicating protein instability. A singl e mutation, E241K, which homology modeling indicated would restore a f avorable charge pair interaction between K241 in helix G and E288 in h elix I, led to successful expression of this chimera that exhibited a K-S < 10 mu M for SFZ, Systematic replacement of the remaining differi ng amino acids revealed that two amino acid substitutions in 2C19 (N28 6S, I289N) confer high-affinity SFZ binding (K-S < 5 mu M). When combi ned with a third substitution, E241K, the resulting 2C19 triple mutant exhibited a high cataltyic efficiency for warfarin metabolism with th e relaxed stereo- and regiospecificity of 2C19 and a lower KM for (S)- warfarin metabolism (<10 mu M) typical of 2C9.