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  • Title: Affinity labeling identifies histidine at the active site of human placental 3 beta-hydroxysteroid dehydrogenase and steroid 5-->4-ene-isomerase.
    Author: Strickler RC, Thomas JL.
    Journal: Am J Obstet Gynecol; 1993 Apr; 168(4):1216-21; discussion 1221-2. PubMed ID: 8475968.
    Abstract:
    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. STUDY DESIGN: 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. CONCLUSION: 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.
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