Human saturated steroid 6alpha-hydroxylase.
This study was conducted to evaluate further the reaction catalyzed by the saturated steroid 6alpha-hydroxylase of extrahepatic human tissues. Progesterone and 5alpha-dihydroprogesterone (5alpha-DHP) are plasma-borne precursors of 5alpha-pregnan-3alpha-ol-20-one, an anxiolytic/anesthetic steroid, and 5alpha-pregnan-3beta-ol-20-one in extrahepatic human tissues. These two steroids are metabolized further by a saturated steroid 6alpha-hydroxylase enzyme(s) that is distinct from the cytochrome P450 6alpha-hydroxylase that catalyzes the 6alpha-hydroxylation of delta4-3-ketosteroids such as progesterone, cortisol, and testosterone. Products of this saturated steroid 6alpha-hydroxylase, viz. 3beta/alpha,6alpha-dihydroxy-5alpha-pregnan-20-ones, are major radiolabeled urinary metabolites (excreted as glucuronosides) of i.v. administered tritium-labeled 5alpha-DHP in women and men. T47-D human breast cancer cells, which are rich in saturated steroid 6alpha-hydroxylase activity, were used as the enzyme source in this study. The greatest total and the highest specific activity of saturated steroid 6alpha-hydroxylase were localized in microsome-enriched preparations; enzyme activity was linear with incubation time up to 30 min and with microsome-enriched tissue protein concentrations between 0.05-0.5 mg/mL incubation mixture. The velocity of the reaction was similar in incubations in which the pH was varied from 6.0-8.0, and NADH and NADPH were equally effective in supporting the 6alpha-hydroxylation of 5alpha-pregnan-3beta-ol-20-one and 5alpha-pregnan-3alpha-ol-20-one. The more efficient substrates for this enzyme were 5alpha-pregnan-3beta-ol-20-one and 5alpha-pregnan-3alpha-ol-20-one, and the apparent Km (approximately 3.5 micromol/L) and maximum velocity (approximately 150 pmol/min x mg microsome-enriched protein) for these two substrates were indistinguishable. 5alpha-Androstane-3beta,17beta-diol was less efficiently 6alpha-hydroxylated, and 5alpha-androstane-3alpha,17beta-diol was an inefficient substrate. The addition of a variety of inhibitors of cytochrome P450 monooxygenases to the incubation mixtures did not diminish significantly the 6alpha-hydroxylation of 5alpha-pregnan-3beta-ol-20-one, findings consistent with those of other investigators who suggested that human saturated steroid 6alpha-hydroxylase (of human prostate) is not a cytochrome P450.
Dombroski
R
R
Department of Obstetrics-Gynecology, University of Texas Southwestern Medical School, Dallas 75235, USA.
Casey
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ML
Macdonald
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PC
D001943:Breast Neoplasms / Q000201:enzymology / Q000648:ultrastructure; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006900:Hydroxylation; D007700:Kinetics; D008297:Male; D008861:Microsomes / Q000201:enzymology; D009243:NAD / Q000494:pharmacology; D009249:NADP / Q000494:pharmacology; D011280:Pregnanolone / Q000378:metabolism*; D013250:Steroid Hydroxylases / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D009243:NAD; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D011280:Pregnanolone; D013250:Steroid Hydroxylases; C105690:saturated steroid 6-alpha-hydroxylase