Purification of human placental estradiol 17 beta-dehydrogenase study of the steroid-binding site.
Human placental estradiol 17 beta-dehydrogenase has been purified by affinity chromatography. The purified enzyme is homogenous by polyacrylamide-gel electrophoresis. To study topography of the steroid-binding site, 16 alpha-bromoacetoxyestradiol 3-methyl ether was synthesized with estriol 3-methyl ether, bromoacetic acid, or [2-3H] bromoacetic acid and dicyclohexylcarbodiimide. The steroid alkylates cysteine, histidine, methionine, and tryptophan under physiologic conditions. Being a substrate of the enzyme, it must bind at the steroid-binding site. The steroid inactivates the enzyme in a time-dependent, irreversible manner. Inactivation of the enzyme by excess 16 alpha-bromoacetoxyestradiol 3-methyl ether follows pseudo--first-order kinetics with t1/2 = 1.5 hours. Amino acid analysis reveals that a histidyl residue is carboxymethylated. Estradiol-17 beta slows inactivation; 2-mercaptoethanol stops it. Previous studies have shown a histidyl residue also present at the catalytic region of the active site of 20 beta-hydroxysteroid dehydrogenase from Streptomyces hydrogenans. It is tempting to consider that these histidyl residues may be an essential component for the dehydrogenation of the steroid substrates.
D001665:Binding Sites; D002846:Chromatography, Affinity / Q000379:methods; D003545:Cysteine / Q000378:metabolism; D004960:Estradiol Dehydrogenases / Q000302:isolation & purification* / Q000378:metabolism; D005260:Female; D006639:Histidine / Q000378:metabolism; D006801:Humans; D006913:Hydroxysteroid Dehydrogenases / Q000302:isolation & purification*; D008715:Methionine / Q000378:metabolism; D010920:Placenta / Q000201:enzymology* / Q000378:metabolism; D011247:Pregnancy; D011956:Receptors, Cell Surface; D014364:Tryptophan / Q000378:metabolism
D011956:Receptors, Cell Surface; D006639:Histidine; D014364:Tryptophan; D008715:Methionine; D006913:Hydroxysteroid Dehydrogenases; D004960:Estradiol Dehydrogenases; D003545:Cysteine