Dual activity at an enzyme active site: 3.beta.,20.alpha.-hydroxysteroid oxidoreductase from fetal blood

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Abstract
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 a single-stranded polypeptide with a MW of 55,000 .+-. 1000 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 .mu.M; Vmax = 2.4 nmol min-1 (nmol of enzyme)-1] and 20.alpha. [Km = 2.5 .mu.M; Vmax = 2.4 nmol min-1 (nmol of enzyme)-1] reductase activities. 17.beta.-Hydroxy-5.alpha.-androstan-3-one competitively inhibited (inhibition constant = 35 .mu.M) 20.alpha. reduction of progesterone. Incubating 3.beta.,20.alpha.-HSD with 19-nortestosterone 17-bromoacetate at pH 7.0 and 25.degree. C caused simultaneous, time-dependent, and irreversible losses of 3.beta. and 20.alpha. activities by a 1st-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. The active site of 3.beta.,20.alpha.-HSD apparently contains 3.beta. and 20.alpha. dual activity.