Structure of the steroid-binding site of human placental estradiol-17beta-dehydrogenase.

We recently demonstrated that human placental estradiol-17beta-dehydrogenase possesses a histidyl residue in the catalytic region of the active site by affinity-labeling studies with 16alpha-bromoacetoxyestradiol-3-methyl ether. We now report the synthesis of 12beta-bromoacetoxy-4-estrene-3,17-dione and its use in affinity labeling of the enzyme. The steroid was synthesized by incubation of 4-estrene-3,17-dione with Colletotrichum gloesporioides. The product was recrystallized from ethanol and structure assured by IR and NMR spectroscopy. The steroid is a substrate, which indicates that it binds at the active site. When the enzyme is incubated with a 150-fold molar excess of 12beta-bromoacetoxy-4-estrene-3,17-dione in potassium phosphate buffer at pH 7.0, the enzyme is inactivated in a time-dependent, irreversible manner. Inactivation follows pseudo-first-order kinetics with a half life of 18 hours. Analysis of a hydrolysate of the enzyme after inactivation with 12beta-bromo[2'-3H]acetoxy-4-estrene-3,17-dione reveals tritiated 1-, 3-, and 1,3-dicarboxymethylhistidine. The affinity labeling of a histidyl enzyme residue by both 16alpha- and 12beta-bromoacetoxy steroids localizes that residue near the point of catalysis and suggests that it may participate in the catalytic event.