The Importance of SRS-1 Residues in Catalytic Specificity of Human Cytochrome P450 3A4

The structural basis for the regioselective hydroxylation of Δ-4-3-ketosteroids by human CYP3A4 was investigated. Prior studies had suggested that the chemical reactivity of the allylic 6β-position might have a greater influence than steric constraints by the enzyme. Six highly conserved CYP3A residues from substrate recognition site 1 were examined by site-directed mutagenesis. F102A and A117L showed no spectrally detectable P450. V101G and T103A exhibited a wild-type progesterone metabolite profile. Of five mutants 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 2β-OH rather than 6β-OH progesterone or testosterone as the major metabolite. Kinetic analysis showed S50 values similar to wild-type for S119A (progesterone) and S119V (testosterone), whereas the Vmax values for 2β-hydroxysteroid formation were increased in both cases. All four mutants exhibited an altered product profile for 7-hexoxycoumarin side-chain hydroxylation, whereas the stimulation of steroid hydroxylation by α-naphthoflavone was similar to the wild-type. The results indicate that the highly conserved residue S119 is a key determinant of CYP3A4 specificity and reveal an important role of the active site topology in steroid 6β-hydroxylation.