Microsomal 17β-hydroxysteroid dehydrogenase of guinea pig liver: Substrate and inhibitor specificity and effects of pH on steroid-enzyme interaction

Various C 18 and C 19 steroids and some nonsteroidal compounds were assayed at pH 9.0 as substrates or inhibitors of microsomal 17β-hydroxysteroid dehydrogenase of guinea pig liver. C 18 and C 19 steroids with a 17β-hydroxyl group had Michaelis constants ( K m ) or inhibition constants ( K i ), competitive with testosterone, of 5.8 to 59.0 μ m. Initial velocity estimates as a function of substrate concentration with combined substrates indicated that both C 18 and C 19 steroids were bound at the same active site. When pH was varied the K m values for testosterone and 19-nortestosterone increased below pH 7.5 while the K m for NAD + and the K i for androstenedione were relatively unchanged. Based on the reactivity of both C 18 and C 19 17β-hydroxysteroids as substrates and the lack of reactivity of cyclopentanol and cyclohexanol as substrates as well as their low affinities as inhibitors, it is proposed that the geometry of the steroid binding site is such as to allow for compounds such as C 18 and C 19 17β-hydroxysteroids to bind but that a minimum size is required for tight binding, consistent with previous observations on human placental 17β-hydroxysteroid dehydrogenase and estrogen receptor. And, as with these latter proteins, the presence of a hydroxyl group at C(17) appears to influence the mode of steroid recognition.