The importance of SRS-1 residues in catalytic specificity of human cytochrome p450 3A4

The structural basis for the regioselective hydroxylation of Delta-4-3-keto steroids by human CYP3A4 was investigated. Prior studies had suggested that the chemical reactivity of the allylic 6 beta-position might have a greate r influence than steric constraints by the enzyme. Six highly conserved CYP 3A residues from substrate recognition site 1 were examined by site-directe d mutagenesis. F102A and A117L showed no spectrally detectable P450. V101G and T103A exhibited a wild-type progesterone metabolite profile. Of five mu tants at residue N104, only N104D yielded holoenzyme and exhibited the same steroid metabolite profile as wild-type. Of four mutants at position S119 (A, L, T, V), the three hydrophobic ones produced 2 beta-OH rather than 6 b eta-OH progesterone or testosterone as the major metabolite. Kinetic analys is showed S-50 values similar to wild-type for S119A (progesterone) and S11 9V (testosterone), whereas the V-max values for 2 beta-hydroxysteroid forma tion were increased in both cases. All four mutants exhibited an altered pr oduct profile for 7-hexoxycoumarin side-chain hydroxylation, whereas the st imulation of steroid hydroxylation by alpha-naphthoflavone was similar to t he wild-type. The results indicate that the highly conserved residue S119 i s a hey determinant of CYP3A4 specificity and reveal an important role of t he active site topology in steroid 6 beta-hydroxylation. (C) 2000 Academic Press.