Human placental estradiol 17 beta-dehydrogenase. Identification of a single histidine residue affinity-labeled by both 3-bromoacetoxyestrone and 12 beta-bromoacetoxy-4-estrene-3,17-dione.
Human placental estradiol 17 beta-dehydrogenase (EC 1.1.1.62) was affinity-labeled at pH 6.3 by 3-bromo[2'-14C]acetoxyestrone and 12 beta-bromo-[2'-14C] acetoxy-4-estrene-3,17-dione (both are substrates) in separate incubations. The affinity-alkylated enzyme samples were then treated separately as described below. Amino acid compositions of both samples revealed radioactive 3-carboxymethylhistidine. Tryptic digests of each sample were prepared, applied to Sephadex G-50, and 3-carboxymethylhistidine-bearing fractions identified. These peptides were further purified by cation exchange chromatography, gel filtration, and paper electrophoresis. The purified, 3-carboxymethylhistidine-bearing peptides labeled by the two steroids had identical electrophoretic mobilities at pH 6.5, 3.5, and 1.9. The amino acid sequence of the radioactive peptide alkylated by 3-bromo[2'-14C]acetoxyesterone was determined as: Leu-Ala-3-[14C]CmHis-Ser-Lys. The smaller quantity of peptide obtained from the inactivation with 12 beta-bromo[2'-14C]acetoxy-4-estrene-3,17-dione precluded the determination of its complete sequence. However, the first 3 residues were found to be Leu-Ala-3-[14C]CmHis and the amino acid composition showed that serine and lysine were also present. It is concluded that the steroid-binding site of human placental estradiol 17 beta-dehydrogenase contains a histidine residue which proximates the upper A-ring region of the steroid as it undergoes the reversible binding step.
D015067:17-Hydroxysteroid Dehydrogenases / Q000378:metabolism*; D000345:Affinity Labels / Q000494:pharmacology*; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D002250:Carbon Radioisotopes / Q000032:analysis; D004960:Estradiol Dehydrogenases / Q000378:metabolism*; D004963:Estrenes / Q000494:pharmacology*; D004970:Estrone / Q000031:analogs & derivatives*; D005260:Female; D006639:Histidine; D006801:Humans; D006894:Hydroxyestrones / Q000494:pharmacology*; D010446:Peptide Fragments / Q000032:analysis; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D014357:Trypsin
D000345:Affinity Labels; D000596:Amino Acids; D002250:Carbon Radioisotopes; D004963:Estrenes; D006894:Hydroxyestrones; D010446:Peptide Fragments; D004970:Estrone; D006639:Histidine; C015720:12 beta-bromoacetoxy-4-estrene-3,17-dione; C034312:estrone 3-bromoacetate; D015067:17-Hydroxysteroid Dehydrogenases; D004960:Estradiol Dehydrogenases; D014357:Trypsin