Site-directed mutagenesis identifies amino acid residues associated with the dehydrogenase and isomerase activities of human type I (placental) 3β- hydroxysteroid dehydrogenase/isomerase

3β-Hydroxysteroid dehydrogenase/steroid Δ(5→4)-isomerase (3β- HSD/isomerase) was expressed by baculovirus in Spodoptera fungiperda (Sf9) insect cells from cDNA sequences encoding human wild-type I (placental) and the human type I mutants - H261R, Y253F and Y253,254F. Western blots of SDS- polyacrylamide gels showed that the baculovirus-infected Sf9 cells expressed the immunoreactive wild-type, H261R, Y253F or Y253,254F protein that co- migrated with purified placental 3β-HSD/isomerase (monomeric M(r)=42,000 Da). The wild-type, H261R and Y253F enzymes were each purified as a single, homogeneous protein from a suspension of the Sf9 cells (5.0 1). In kinetic studies with purified enzyme, the H261R mutant enzyme had no 3β-HSD activity, whereas the K(m) and V(max) values of the isomerase substrate were similar to the 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 androstenedione by the Y253F isomerase activity was 7.0-fold less than the mean V(max) (620 nmol/min/mg) measured for the isomerase activity of the wild-type and native placental enzymes. In microsomal preparations, isomerase activity was completely abolished in the Y253,254F mutant enzyme, but Y253,254F had 45% of the 3β-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 3β-HSD activity. The 3β-HSD activities of the Y253F, Y253,254F and wild-type enzymes reduced NAD⁺ with similar kinetic values. Although NADH activated the isomerase activities of the H261R and wild-type enzymes with similar kinetics, the activation of the isomerase activity of H261R by NAD⁺ was dramatically decreased. Based on these kinetic measurements, His²⁶¹ appears to be a critical amino acid residue for the 3β-HSD activity, and Tyr²⁵³ or Tyr²⁵⁴ participates in the isomerase activity of human type I (placental) enzyme.