SITE-DIRECTED MUTAGENESIS IDENTIFIES AMINO-ACID-RESIDUES ASSOCIATED WITH THE DEHYDROGENASE AND ISOMERASE ACTIVITIES OF HUMAN TYPE-I (PLACENTAL) 3-BETA-HYDROXYSTEROID DEHYDROGENASE ISOMERASE/

3 beta-Hydroxysteroid dehydrogenase/steroid Delta(5 --> 4)-isomerase ( 3 beta-HSD/isomerase) was expressed by baculovirus in Spodoptera fungi perda (Sf9) insect cells from cDNA sequences encoding human wild-type I (placental) and the human type I mutants - H261R, Y253F and Y253,254 F. Western blots of SDS-polyacrylamide gels showed that the baculoviru s-infected Sf9 cells expressed the immunoreactive wild-type, H261R, Y2 53F or Y253,254F protein that co-migrated with purified placental 3 be ta-HSD/isomerase (monomeric M-r=42,000 Da). The wild-type, H261R and Y 253F enzymes were each purified as a single, homogeneous protein from a suspension of the Sf9 cells (5.0 1). In kinetic studies with purifie d enzyme, the H261R mutant enzyme had no 3 beta-HSD activity, whereas the K-m and V-max values of the isomerase substrate were similar to th e values obtained with the wild-type and native enzymes. The V-max (88 nmol/min/mg) for the conversion of 5-androstene-3,17-dione to androst enedione by the Y253F isomerase activity was 7.0-fold less than the me an V-max (620 nmol/min/mg) measured for the isomerase activity of the wild-type and native placental enzymes. In microsomal preparations, is omerase activity was completely abolished in the Y253,254F mutant enzy me, but Y253,254F had 45% of the 3 beta-HSD activity of the wild-type enzyme. In contrast, the purified Y253F, wild-type and native enzymes had similar V-max values for substrate oxidation by the 3 beta-HSD act ivity. The 3 beta-HSD activities of the Y253F, Y253,254F and wild-type enzymes reduced NAD(+) with similar kinetic values. Although NADH act ivated the isomerase activities of the H261R and wildtype enzymes with similar kinetics, the activation of the isomerase activity of H261R b y NAD(+) was dramatically decreased. Based on these kinetic measuremen ts, His(261) appears to be a critical amino acid residue for the 3 bet a-HSD activity, and Tyr(253) Or Tyr(254) participates in the isomerase activity of human type I (placental) enzyme. (C) 1998 Elsevier Scienc e Ltd. All rights reserved.