Affinity radiolabeling identifies peptides associated with the isomerase activity of human type I (placental) 3beta-hydroxysteroid dehydrogenase/isomerase.

3beta-Hydroxysteroid dehydrogenase and steroid Delta5-->4-isomerase (3beta-HSD/isomerase) were purified as a single protein from human term placenta. The affinity alkylator, 5,10-secoestr-4-yne-3,10, 17-trione (secosteroid), was incubated with the purified enzyme (30/1 secosteroid/enzyme molar ratio) to produce an 80% loss of initial isomerase activity over 90 min in a time-dependent, irreversible manner. The secosteroid inactivated 3beta-HSD by only 20% during the same 90 min. Incubations containing the isomerase substrate steroid, 5-androstene-3,17-dione, completely protected the isomerase activity from inactivation by the secosteroid and did not slow the inactivation of 3beta-HSD. The enzyme containing covalently bound steroid was separated from unreacted secosteroid by reversed phase HPLC. Ketones on the protein-bound secosteroid were radiolabeled by reduction with sodium boro[3H]hydride (specific radioactivity 50 microCi/micromol for the transferred tritium). After removal of the unreacted sodium boro[3H]hydride, the affinity-radiolabeled enzyme was digested with trypsin-TPCK, and the peptides were isolated by reversed phase HPLC. The radiolabeled peptide fractions were sequenced. The secosteroid alkylated three tryptic peptides: 251GQFYYISDDTPHQSYDNLNYTLSK274, tritiated His262; 176NGGTLYTCALR186, tritiated Cys183; and 353TVEWVGSLVDR363, tritiated Trp356. Coincubation with the isomerase substrate blocked the labeling of these three peptides and shifted the alkylation by secosteroid to a single tryptic peptide (135EIIQNGHEEEPLENTWPAPYPHSK159, tritiated His142). Using substrate protection to validate specificity, the affinity labeling secosteroid has identified peptides in the enzyme that are associated with isomerase activity.