The steroids, 17β-[1(R)-1-hydroxy-2-propynyl]androst-4-en-3-one (α-HPA) and 17β-(1-oxo-2-propynyl)-androst-4-en-3-one (OPA), were used to investigate the 17β-estradiol dehydrogenase and 20α-hydroxysteroid dehydrogenase activities which co-exist in the homogeneous enzyme purified from human placental cytosol. OPA is a substrate (with NADH, apparent K(m) =166 μM; V(max)=1.51 μmol/min/mg) and a very rapid irreversible affinity alkylator (K(I)=190 μM; k3 =9.47 x 10-2 s-1 determined without cofactor) which causes simultaneous inactivation of the 17β- and 20α-activities in a time-dependent manner which follows pseudo-first order kinetics (t(1/2)=0.45 min, pH 9.2, and 50 μM; t(1/2)= 5.2 min, pH 7.0, and 50 μM). Enzyme substrates, estrone, estradiol-17β, progesterone, and 20α-dihydroprogesterone, protect against inactivation by OPA. α-HPA does not inactivate the enzyme in the absence of NAD+. However, in the presence of NAD+, α-HPA is a poor substrate (K(m)=435 μM; V(max) 0.04 μmol/min/mg). Consequently, when α-HPA is enzymatically oxidized in the presence of excess NAD+, identical, time-dependent, irreversible inactivation of both the 17β- and 20α-activities results with half-times of 138 min (pH 9.2; 100 μM) and 500 min (pH 7.0; 50 μM). The β-isomer, 17β[(1S)-1-hydroxy-2-propynyl]androst-4-en-3-one, is not oxidized by and, therefore, does not inactivate the enzyme. The simultaneous inactivation of both the major 17β-estradiol dehydrogenase and 20α-hydroxysteroid dehydrogenase activities by OPA and by enzymatic oxidation of α-HPA clearly demonstrates the bifunctional activity of the single enzyme-active site.
|Number of pages||4|
|Journal||Journal of Biological Chemistry|
|State||Published - 1982|