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.
Thomas
J L
JL
Department of Obstetrics and Gynecology, Washington University School of Medicine, St. Louis, Missouri 63110, USA. Thomas_J@kids.wustl.edu
Evans
B W
BW
Strickler
R C
RC
D000345:Affinity Labels / Q000378:metabolism*; D000478:Alkylation; D000595:Amino Acid Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D004963:Estrenes / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010449:Peptide Mapping; D010920:Placenta / Q000201:enzymology*; D011375:Progesterone Reductase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013251:Steroid Isomerases / Q000037:antagonists & inhibitors / Q000378:metabolism*
C021425:3 beta-hydroxysteroid oxidoreductase-delta(5) 3-ketosteroid isomerase; D000345:Affinity Labels; D004963:Estrenes; D009097:Multienzyme Complexes; C024783:5,10-secoestr-5-yne-3,10,17-trione; D011375:Progesterone Reductase; D013251:Steroid Isomerases