Kinetics of inhibition of estrogen 2-hydroxylase by various haloestrogens

Inhibitors of estrogen 2-hydroxylase can be utilized in studying the kinetics of this cytochrome P450 enzyme complex and in elucidating the structural requirements of the active site. The conversion of estrogens to 2-hydroxyestrogens in rat liver microsomal preparations was examined using two radiotracer assays, the conversion of [4-14C]-estradiol to [4-14C]-2-hydroxyestradiol and the release of 3H2O from [2-3H]-estradiol. Using the microsomal fraction from male rat liver, the apparent K/sub m/ for the substrate estradiol was 2.2 microM. Competitive inhibition was observed for 2-halo- and 2,4-haloestrogens (apparent K/sub i/'s of 1.6 to 3.7 microM), while 4-haloestrogens did not produce normal inhibition patterns. Employing female rat liver microsomes in which nonclassical enzyme kinetics was observed, the synthetic steroids increased the sigmoidal character of the velocity curve. Multiple inhibition studies with 2-haloestrogens and 4-haloestrogens with the male rat liver microsomal fraction indicated that these compounds are mutually exclusive inhibitors of the 2-hydroxylase activity