HYDROXYSTEROID DEHYDROGENASES 449 



This observation was extended to a number of estra-l,3,5-trienes, most of 

 which are inhibitory to the oxidation of testosterone and are not themselves 

 attacked (Marcus and Talalay, 1955). The most potent inhibitors are the 

 following derivatives of estratriene: -3,17a-diol (a-estradiol), -3,16«-diol; 

 -3,16a,17/5-triol (estriol); -3-ol; -3,17/?-diol-16-one; and -3,16/3-diol. It ap- 

 pears that the aromaticity of ring A combined with the 3-OH group re- 

 sults in strong binding. The 17-ols are not inhibitory although there are 

 enzyme regions for the oxidation of either 3-OH or 17-OH groups in other 

 ring systems. The total ring system is not necessary since dieth/lstilbestrol 

 and hexestrol are potent inhibitors. The K/s for most of the effective in- 

 hibitors are around 0.001-0.01 mM corresponding to over-all interaction 

 energies of 7-8.5 kcal/mole, implying a rather close fit over the surface of 

 the molecules and a summation of dispersion and polarization forces. It is 

 possible that the high polarizability of the aromatic ring A is important in 

 augmenting binding, this ring overlying some ionic group on the enzyme, 

 and the 3-OH interacts to form a hydrogen bond. The a,^ specificity indi- 

 cates that the steroids attach to the enzyme by their "rear" surfaces. The 

 cf-hydroxysteroid dehydrogenase is not inhibited so readily by the estra- 

 trienes as is the (3 enzyme (Talalay and Marcus, 1956). It was stated that 

 the inhibitions are neither exactly competitive nor noncompetitive, but no 

 data or plots were given, nor was the exact experimental procedure des- 

 cribed, so that it is impossible evaluate the nature of the inhibitions. 



The most potent inhibitor of the Pseudomonas /?-hydroxysteroid dehydro- 

 genase yet found is the noncompetitive 2-hydroxymethylene-17a-methylan- 

 drostan-17/5-ol-3-one {K, = 0.0003 mM), although the competitive 4,4-di- 

 methyl-17/?-hydroxyandrost-5-eno(3,2-c)pyrazole {K^ = 0.0005 mM) is al- 

 most as active (Ferrari and Arnold, 1963 a, b). The inhibitions by these and 

 simpler steroids are dependent on the pH; e.g.. diethylstilbestrol is approxi- 

 mately 20 times as effective at pH 8.5 than at pH 5.5. Since it is unlikely 

 that the phenolic groups would ionize in this range (pK^ for diethylstilbes- 

 trol is 12.2), this implies the ionization of enzyme groups at or near the 

 active site. This emphasizes the importance of polarization of the aromatic 

 rings by anionic groups on the enzyme in determining the tightness of 

 binding. 



The J^- and J*-dehydrogenases which introduce unsaturation into ring 

 A of the 3-ketosteroids at the 1- and 4-positions, respectively, are inducible 

 enzymes in P. testosteroni, and both are inhibited quite potently by estrone 

 (Levy and Talalay, 1959). The J*-3-ketosteroid reductase (5a) of rat liver 

 microsomes, which catalyzes the hydrogenation of the 4-5 double bond, is 

 a NADPH-requiring enzyme acting on cortisone, Cortisol, desoxycorticoste- 

 rone, and related steroids (McGuire et al., 1960). It is competitively inhi- 

 bited by a variety of less substituted steroids, such as androst-l-ene-3,17- 

 dione and 5a-androstane-3,17-dione; the 5/?-androstane-3,17-dione is, how- 



