Mechanism of 4-ene-steroid 5 alpha-reductase proton transfer in androgen target tissues

The conversion of testosterone to 5 alpha-dihydrotestosterone, catalysed by 4-ene-steroid 5 alpha-reductase (3-oxo-5 alpha-steroid: NADP+ 4-ene-oxidoreductase EC 1.3.1.22) requires NADPH. In the present study, the role of flavins and Co-enzyme Q in this proton transfer was investigated for the first time in any male androgen target tissue. Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) inhibited epididymal nuclear 4-ene-steroid 5 alpha-reductase activity non-competitively with respect to the substrate testosterone. However, neither the oxidized nor reduced forms of Co-enzyme Q affected the Kmapp or the Vmaxapp and the reduced form was unable to support catalytic activity in the absence of NADPH. Further investigation of the effects of flavins revealed that the inhibition was caused by an elevation of NADP+ in the incubations and that the incorporation of a NADPH generating system abolished the inhibition. Therefore, neither flavins nor Co-enzyme Q directly affected the 4-ene-steroid 5 alpha-reductase activity. Further evidence to support this conclusion was obtained when several inhibitors of electron transfer reactions failed to inhibit 4-ene-steroid 5 alpha-reductases from rat epididymides, prostate and seminal vesicles. These findings show that, in male rat androgen target tissues, the conversion of testosterone to 5 alpha-dihydrotestosterone does not require intermediates of electron transfer reactions. We propose that the reduction proceeds by the direct transfer of protons from NADPH to testosterone.