Fetal lamb 3 beta, 20 alpha-hydroxysteroid oxidoreductase: dual activity at the same active site examined by affinity labeling with 16 alpha-(bromo[2'-14C]acetoxy)progesterone.
3 beta,20 alpha-Hydroxysteroid oxidoreductase was purified to homogeneity from fetal lamb erythrocytes. The Mr 35,000 enzyme utilizes NADPH and reduces progesterone to 4-pregnen-20 alpha-ol-3-one [Km = 30.8 microM and Vmax = 0.7 nmol min-1 (nmol of enzyme)-1] and 5 alpha-dihydrotestosterone to 5 alpha-androstane-3 beta, 17 beta-diol [Km = 74 microM and Vmax = 1.3 nmol min-1 (nmol of enzyme)-1]. 5 alpha-Dihydrotestosterone competitively inhibits (Ki = 102 microM) 20 alpha-reductase activity, suggesting that both substrates may be reduced at the same active site. 16 alpha-(Bromoacetoxy)progesterone competitively inhibits 3 beta- and 20 alpha-reductase activities and also causes time-dependent and irreversible losses of both 3 beta-reductase and 20 alpha-reductase activities with the same pseudo-first order kinetic t1/2 value of 75 min. Progesterone and 5 alpha-dihydrotestosterone protect the enzyme against loss of the two reductase activities presumably by competing with the affinity alkylating steroid for the active site of 3 beta,20 alpha-hydroxysteroid oxidoreductase. 16 alpha-(Bromo[2'-14C]acetoxy) progesterone radiolabels the active site of 3 beta,20 alpha-hydroxysteroid oxidoreductase wherein 1 mol of steroid completely inactivates 1 mol of enzyme with complete loss of both reductase activities. Hydrolysis of the 14C-labeled enzyme with 6 N HCl at 110 degrees C and analysis of the amino acid hydrolysate identified predominantly N pi-(carboxy[2'-14C]methyl)histidine [His(pi-CM)].(ABSTRACT TRUNCATED AT 250 WORDS)
Chen
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Division of Reproductive Biology, Washington University School of Medicine, St. Louis, Missouri 63110.
Rosik
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Nancarrow
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Sweet
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D000345:Affinity Labels; D000478:Alkylation; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D005312:Fetal Blood / Q000201:enzymology*; D006908:Hydroxyprogesterones / Q000493:pharmacokinetics*; D006913:Hydroxysteroid Dehydrogenases / Q000037:antagonists & inhibitors / Q000097:blood*; D009682:Magnetic Resonance Spectroscopy; D011247:Pregnancy; D012756:Sheep
D000345:Affinity Labels; D000596:Amino Acids; D006908:Hydroxyprogesterones; C000311:16 alpha-bromoacetoxyprogesterone; D006913:Hydroxysteroid Dehydrogenases; C036177:3 beta,20 alpha-hydroxysteroid oxidoreductase