Affinity alkylation of bacterial delta 5-3-ketosteroid isomerase. Identification of the amino acid modified by steroidal 17 beta-oxiranes.
The two steroidal 17 beta-oxiranes, spiro-17 beta-oxiranyl-delta 4-androsten-3-one (4 beta) and spiro-17 beta-oxiranylestra-1,3,5(10),6,8-pentaene-3-ol (5 beta) are active site-directed irreversible inhibitors of bacterial delta 5-3-ketosteroid isomerase. For each inhibitor, a stoichiometry of one molecule of steroid to one enzyme subunit was found. The inhibited enzyme was denatured and subjected to digestion by trypsin. The tryptic maps show two distinct steroid-bound peptides for both 4 beta- and 5 beta-inhibited isomerase. In each case, the two modified peptides are derived from residues 14 to 45 of the isomerase. Each of the steroid-bound peptides of the 4 beta-inhibited enzyme was subjected to further proteolytic digestion and the site of steroid attachment was found to be Asp-38 in each of the inactivation products. These results are interpreted to indicate that "backwards binding" is an important feature of the binding of steroids to delta 5-3-ketosteroid isomerase.
D000596:Amino Acids / Q000032:analysis; D000736:Androstenes / Q000494:pharmacology; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D004963:Estrenes / Q000494:pharmacology; D007535:Isomerases / Q000037:antagonists & inhibitors*; D010446:Peptide Fragments / Q000032:analysis; D013251:Steroid Isomerases / Q000037:antagonists & inhibitors*; D013997:Time Factors; D014357:Trypsin / Q000378:metabolism
D000596:Amino Acids; D000736:Androstenes; D004963:Estrenes; D010446:Peptide Fragments; C037048:spiro-17-oxiranyl-delta(4)-androsten-3-one; C037049:spiro-17 beta-oxiranylestra-1,3,5(10),6,8-pentaene-3-ol; D014357:Trypsin; D007535:Isomerases; D013251:Steroid Isomerases; C022660:steroid delta-isomerase