Characterization of molecular and catalytic properties of intact and truncated human 17beta-hydroxysteroid dehydrogenase type 2 enzymes: intracellular localization of the wild-type enzyme in the endoplasmic reticulum.

Human 17beta-hydroxysteroid dehydrogenase (17HSD) type 2 is a widely distributed enzyme that primarily converts the highly active 17beta-hydroxysteroids to their inactive keto forms. In the present study, full-length human 17HSD type 2 was localized in the endoplasmic reticulum using a double immunofluorescence labeling technique. As a consequence of its strong membrane interaction, full-length human 17HSD type 2 could not be solubilized as a biologically active form in vitro. However, by deleting the first 29 amino acids from the N-terminus, we were able to purify a catalytically active enzyme from the cytosolic fraction of Sf9 insect cells. Biochemical and catalytic properties of the purified truncated human 17HSD type 2 protein confirm its suitability for structure-function analyses of the enzyme. Both intact and truncated 17HSD type 2 enzymes efficiently catalyzed the oxidation of estradiol, testosterone, dihydrotestosterone, androstenediol, and 20alpha-dihydroprogesterone. The oxidation of estradiol brought about by human 17HSD type 2 was effectively inhibited by several other steroidal compounds, such as 2-hydroxyestradiol, 5beta-androstan-3alpha,17beta-diol, 5alpha-androstan-3alpha,17beta-diol, and 5alpha-androstan-3beta,17beta-diol. The broad substrate specificity of human 17HSD type 2 together with its predominant oxidative activity and intracellular location, as observed in this study, indicate the physiological role of the enzyme to be primarily an inactivator of highly active 17beta-hydroxysteroids.

Site-directed mutagenesis of prostatic acid phosphatase. Catalytically important aspartic acid 258, substrate specificity, and oligomerization.

At the active site of rat prostatic acid phosphatase (rPAP), residue Asp258 is a suitable candidate to act as an acid/base catalyst during phosphoester hydrolysis. It was changed to Asn, Ser, and Ala by site-directed mutagenesis. All these mutants were inactive, indicating that Asp258 may act as a proton donor in catalysis. Tyr123 and Arg127 residues, located at the entrance of the active site surface in rPAP, are likely to be responsible for the substrate specificity of the enzyme. The corresponding residues in lysosomal acid phosphatase (LAP) are Lys and Gly. In order to clarify the roles of the Tyr123 and Arg127 residues, lysosomal type rPAP mutants (Y123K, R127G and Y123K,R127G) were generated. Sensitivity of Y123K,R127G to tartrate inhibition was similar to that observed in the case of LAP, indicating that these residues might be responsible for differences in substrate specificity between the enzymes of prostatic and lysosomal origin. However, unlike human LAP, the lysosomal type mutants hydrolyzed the suggested PAP-specific substrates, phosphocreatine and phosphocholine, showing that Tyr123 and Arg127 are not the only residues contributing to the substrate specificity of rPAP. The residues Trp106 and His112 appeared to be important in the dimerization of rPAP. Oligomerization mutants (W106E, H112D and W106E,H112D) existed in a monomeric form without catalytic activity or a tartrate binding ability.