Isolation of histidyl peptides of the steroid-binding site of human placental estradiol 17 beta-dehydrogenase.
Human placental estradiol 17 beta-dehydrogenase (E.C. 1.1.1.62) was inactivated at pH 6.3 by 3-bromo [2'-14C] acetoxy-1,3,5(10) estratrien-17-one, a know substrate. The affinity-alkylated enzyme was then hydrolyzed by trypsin. Radioactive peptides were initially isolated by gel filtration and identified according to which residue was alkylated. Tryptic peptides containing radioactive 3-carboxymethylhistidyl residues were further purified by cation-exchange chromatography. The population of these peptides varied, depending upon the conditions of enzyme inactivation. With 60 microM 3-bromo[2'-14]acetoxy-1,3,5 (10) estratrien -17-one four major peptides (a,b,c,d) each containing radioactive 3-carboxymethylhistidine, were eluted from the cation-exchange column. The alkylation of all of these peptides was completely suppressed when the enzyme was inactivated in the presence of excess estradiol-17 beta. The presence of equimolar NADPH during incubation greatly enhanced the alkylation of all four peptides. In the presence of NADPH, estradiol-17 beta most significantly decreased the formation of peptide d. Peptide d was the only peptide identified when the concentration of the alkylating steroid was lowered to 6 microM, a value approaching the Km. These observations indicate that peptide d is a histidyl-bearing peptide from the steroid-binding site which proximates the steroid A-ring. They further suggest that with the affinity labeling steroid at higher concentrations other nonspecific, hydrophobic sites on the enzyme are occupied and labeled.
D015067:17-Hydroxysteroid Dehydrogenases / Q000032:analysis*; D001665:Binding Sites; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004960:Estradiol Dehydrogenases / Q000032:analysis* / Q000037:antagonists & inhibitors; D005260:Female; D006639:Histidine; D006801:Humans; D006894:Hydroxyestrones / Q000378:metabolism; D007700:Kinetics; D009249:NADP / Q000494:pharmacology; D010446:Peptide Fragments / Q000302:isolation & purification*; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D014357:Trypsin / Q000378:metabolism
D006894:Hydroxyestrones; D010446:Peptide Fragments; D006639:Histidine; D009249:NADP; C034312:estrone 3-bromoacetate; D015067:17-Hydroxysteroid Dehydrogenases; D004960:Estradiol Dehydrogenases; D014357:Trypsin