Dual activity at an enzyme active site: 3 beta,20 alpha-hydroxysteroid oxidoreductase from fetal blood.
An enzyme exhibiting both 3 beta and 20 alpha steroid reductase activities from calf fetal red blood cells was purified to homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 3 beta,20 alpha-Hydroxysteroid oxidoreductase (3 beta,20 alpha-HSD) was found to be a single-stranded polypeptide with a molecular weight of 55 000 +/- 1 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Sephadex G-100 chromatography. The amino acid composition of 3 beta,20 alpha-HSD was obtained. 17 beta-Hydroxy-5 alpha-androstan-3-one and progesterone were substrates for the enzyme's 3 beta and 20 alpha reductase activities, respectively, which required NADPH for both 3 beta [Km = 9.4 microM; Vmax = 2.4 nmol min-1 (nmol of enzyme)-1] and 20 alpha [Km = 2.5 microM; Vmax = 2.4 nmol min-1 (nmol of enzyme)-1] reductase activities. 17 beta-Hydroxy-5 alpha-androstan-3-one competitively inhibited (Ki = 35 microM) 20 alpha reduction of progesterone. Incubating 3 beta,20 alpha-HSD with 19-nortestosterone 17-bromoacetate at pH 7.0 and 25 degrees C caused simultaneous, time-dependent, and irreversible losses of 3 beta and 20 alpha activities by a first-order kinetic process. Similar incubations with either of the 3 beta or 20 alpha substrates present at concentrations equal to their respective Km values practically doubled the time required for loss of 3 beta and 20 alpha enzyme activities. These data lead us to conclude that the active site of 3 beta,20 alpha-HSD contains 3 beta and 20 alpha dual activity.
D000345:Affinity Labels / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002846:Chromatography, Affinity; D005260:Female; D005312:Fetal Blood / Q000201:enzymology*; D006913:Hydroxysteroid Dehydrogenases / Q000097:blood* / Q000302:isolation & purification; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009277:Nandrolone / Q000031:analogs & derivatives / Q000494:pharmacology; D011247:Pregnancy
D000345:Affinity Labels; D046911:Macromolecular Substances; C009859:17 beta-bromoacetoxy-19-nortestosterone; D009277:Nandrolone; D006913:Hydroxysteroid Dehydrogenases; C036177:3 beta,20 alpha-hydroxysteroid oxidoreductase