Affinity labeling identifies histidine at the active site of human placental 3 beta-hydroxysteroid dehydrogenase and steroid 5-->4-ene-isomerase.
OBJECTIVE
Our aim was to determine if the multifunction enzyme, 3 beta-hydroxysteroid dehydrogenase and steroid 5-->4-ene-isomerase has one or more active sites to effect dehydrogenase and isomerase activities.


METHODS
This steroid, which we have purified to homogeneity from human placental microsomes, was inactivated by the affinity labeling steroid, 2 alpha-bromo[2'-14C]acetoxyprogesterone. The amino acids that were radioalkylated in the absence and presence of the dehydrogenase substrate pregnenolone were identified.


RESULTS
Pregnenolone completely abolished the inactivation of dehydrogenase. Histidine was localized in the active site of 3 beta-hydroxysteroid dehydrogenase because the radiolabel disappeared from enzyme inactivated in the presence of pregnenolone. Cysteine, a major radiolabeled product (80%) in the absence of pregnenolone, was decreased twofold in incubations that contained pregnenolone. Neither pregnenolone nor the isomerase substrate 5-androstene-3,17-dione protected isomerase from inactivation by the affinity alkylator.


CONCLUSIONS
This observation contradicts coexisting, separate binding sites, one for each activity. Rather, a conformation shift around one binding region prompted by products of the dehydrogenase reaction may create the isomerase activity.
Strickler
R C
RC
Department of Obstetrics and Gynecology, Washington University School of Medicine, St. Louis, MO.
Thomas
J L
JL
D015096:3-Hydroxysteroid Dehydrogenases / Q000737:chemistry*; D000345:Affinity Labels; D000478:Alkylation; D001665:Binding Sites; D001667:Binding, Competitive; D003545:Cysteine / Q000032:analysis; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D006639:Histidine / Q000032:analysis*; D006801:Humans; D006908:Hydroxyprogesterones; D010920:Placenta / Q000201:enzymology*; D011284:Pregnenolone / Q000378:metabolism / Q000494:pharmacology; D013251:Steroid Isomerases / Q000737:chemistry*
D000345:Affinity Labels; D006908:Hydroxyprogesterones; D006639:Histidine; C010871:2-bromoacetoxyprogesterone; D011284:Pregnenolone; D015096:3-Hydroxysteroid Dehydrogenases; D013251:Steroid Isomerases; D003545:Cysteine