Inactivation of human placental 17β,20α-hydroxysteroid dehydrogenase by 16-methylene estrone, an affinity alkylator enzymatically generated from 16-methylene estradiol-17β

The substrate of 16-methylene estra-1,3,5(10)-triene-3,17β-diol (16-methylene estradiol-17β) and its enzyme-generated alkylating product, 3-hydroxy-16-methylene estra-1,3,5(10)-triene-17-one (16-methylene estrone), were synthesized to study the 17β- and 20α-hydroxysteroid dehydrogenase activities which coexist in homogeneous enzyme purified from human placental cytosol. 16-Methylene estradiol, an excellent substrate (K(m) = 8.0 μM; V(max) = 2.8 μmol/mg/min) when enzymatically oxidized to 16-methylene estrone in the presence of NAD⁺ (256 μM), inactivates simultaneously the 17β- and 20α-activities in a time-dependent and irreversible manner following pseudo-first order kinetics (t( 1/2 ) = 1.0 h, 100 μM, pH 9.2). 16-Methylene estradiol does not inactivate the enzyme in the absence of NAD⁺. 16-Methylene estrone (K(m) = 2.7 μM; V(max) = 2.9 μmol/mg/min) is an affinity alkylator (biomolecular rate constant k'₃ = 63.3 liters/mol-s, pH 9.2; K(I) = 261 μM' k₃ = 8.0 x 10^-4 s^-1, pH 7.0) which also simultaneously inhibits both activities in an irreversible time-dependent manner (at 25 μM; t( 1/2 ) = 7.2 min, pH 9.2; t( 1/2 ) = 2.7 h, pH 7.0). Substrates (estradiol-17β, estrone, and progesterone) protect against inhibition of enzyme activity by 16-methylene estrone and 16-methylene estradiol. Affinity radioalkylation studies using 16-methylene [6,7-³H]estrone demonstrate that 1 mol of alkylator binds per mol of inactivated enzyme dimer. Thus, 16-methylene estradiol functions as a unique substrate for the enzymatic generation of a powerful affinity alkylator of 17β,20α-hydroxysteroid dehydrogenase and should be a useful pharmacological tool.