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  • Title: Functional activity of 3beta-hydroxysteroid dehydrogenase/isomerase.
    Author: Mason JI, Naville D, Evans BW, Thomas JL.
    Journal: Endocr Res; 1998; 24(3-4):549-57. PubMed ID: 9888536.
    Abstract:
    3Beta-hydroxysteroid dehydrogenase/steroid delta5-isomerase (3beta-HSD/isomerase) was expressed by baculovirus in Spodoptera fungiperda (Sf9) insect cells from cDNA sequences encoding the human wild-type I (placental) enzyme and the human type I mutant- Y253F. The wild-type and Y253F enzymes were each purified as a single, homogeneous protein from a suspension of the Sf9 cells. Ultraviolet (UV) spectral analyses showed that the wild-type enzyme induced changes in the UV spectrum of the competitive isomerase inhibitor, 19-nortestosterone, and the Y253F mutant did not. The wild-type isomerase required activation by coenzyme to produce the spectral shift. Activation of isomerase by NADH produced a greater change in the 19-nortestosterone spectrum than activation by NAD+. These observations provide direct evidence that Tyr253 functions as the general acid (proton donor) in the isomerase reaction mechanism. Furthermore, the coenzyme-activation profiles support our proposed two-step enzyme mechanism in which NADH produced by the 3beta-HSD activity induces the enzyme to assume the isomerase conformation.
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