Functional activity of 3beta-hydroxysteroid dehydrogenase/isomerase.
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.
Mason
J I
JI
Department of Clinical Biochemistry, Royal Infirmary of Edinburgh NHS Trust, United Kingdom.
Naville
D
D
Evans
B W
BW
Thomas
J L
JL
D000818:Animals; D002460:Cell Line; D004789:Enzyme Activation / Q000502:physiology; D006801:Humans; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D009154:Mutation / Q000502:physiology; D009243:NAD / Q000494:pharmacology; D009277:Nandrolone / Q000737:chemistry / Q000378:metabolism; D011375:Progesterone Reductase / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D013056:Spectrophotometry, Ultraviolet; D018411:Spodoptera / Q000166:cytology; D013251:Steroid Isomerases / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology
C021425:3 beta-hydroxysteroid oxidoreductase-delta(5) 3-ketosteroid isomerase; D009097:Multienzyme Complexes; D009243:NAD; D009277:Nandrolone; D011375:Progesterone Reductase; D013251:Steroid Isomerases