The hydroxysteroid dehydrogenases are a family of widely distributed pyridine nucleotide-linked enzymes which catalyze the freely reversible interconversion of various hydroxy- and ketosteroids. Detailed kinetic studies of the properties of highly purified hydroxysteroid dehydrogenases reveal that these enzymes are saturated with very low concentrations of certain steroids and possess a high degree of structural and steric specificity for their substrates. The capacity of hydroxysteroid dehydrogenases to promote oxidation and reduction reactions between stoichiometric quantities of steroids and pyridine nucleotides did not suggest an explicit functional role for these enzymes in relation to the mechanism of action of steroid hormones. The following events led to the recognition that hydroxysteroid dehydrogenases may under suitable conditions assume the function of transporting hydrogen between pyridine nucleotides in the presence of catalytic concentrations of these steroids (182).

Placental Enzyme

The experiments of Villee and colleagues (reviewed by Villee, C. A.; Hagerman, D. D.; and Joel, P. B. Recent Progress in Hormone Research, 16148, i960) demonstrated that the direct addition of very low concentrations of estradiol-17β or of estrone to slices of human placenta or endometrium enhanced the rate of oxidation of a variety of substrates. Upon further examination of this effect in soluble preparations of human placenta, the stimulatory effect of estrogenic hormones was ascribed to an activation of a DPN-linked isocitric dehydrogenase (Villee, be. cit.). Experiments in this laboratory showed that this hormonal effect could not be demonstrated when the crude placental enzyme system was fractionated by ammonium sulfate precipitation, but it could be fully restored by the addition of catalytic quantities of TPN (182). This finding led to the unequivocal demonstration that the reaction stimulated by estradiol-17β was a transfer of hydrogen from TPNH to DPN (Fig. 20) and was in no special way related to the oxidation of isocitrate (173). The apparent stimulation of a DPN-linked isocitric dehydrogenase could then be understood in terms of the following coupled reactions:

Fig. 20.-Time course of the formation of reduced pyridine nucleotides. The reactions were carried out in 3.0 ml. systems containing 300 µmoles tris pH 7.4; 1 µmole DPN; 1 pinole MnCl2; 1.5 µmole sodium isocitrate; 107 µg- purified rat heart isocitric dehydrogenase; and 1.92 mg. protein of a 0-35 per cent saturated ammonium sulfate fraction of a placental extract. Cuvettes 2 and 4 contained 4 µg- estradioI-17β in 0.01 ml. dioxane initially. TPN (0.023 µmole) was added at 5 minutes to cuvettes 1 and 2. Four µg. estradiol-17β were added to cuvettes 1 and 3 at 120 minutes. All cuvettes received 10 µmoles acctaldehyde in 0.01 ml. at 167 minutes and an excess of yeast alcohol dehydrogenase at 175 minutes. Optical measurements at 340 mµ against a control containing enzyme and buffer. Temp. 25° (182). (Reproduced with permission from the Proceedings of the National Academy of Sciences, Washington, D.C.).

The TPN-linked isocitric dehydrogenase of placenta was unaffected by steroid hormones and could be replaced by any other system capable of reducing TPN (173). These findings were rapidly confirmed by others (Villee, C. A., and Hagerman, D. D. J. Biol. Chem., 233:42, 1958; Hollander, V. P.; Hollander, N.; and Brown, J. D. J. Biol. Chem., 234:1678, 1959).

In the meantime, apparendy unrelated studies on the metabolism of estrogens by placenta had revealed the presence in this tissue of a soluble 17β-hydroxysteroid dehydrogenase which could interconvert estradiol-17β and estrone (Langer, L. J., and Engel, L. L. J. Biol. Chem., 233:583, 1958). This enzyme reacted with TPN and DPN at nearly equal rates:

Estradiol-17β + DPN+ (TPN+) ⇋ estrone + DPNH (TPNH) + H+ .

Further experiments in this laboratory then suggested that the estradiol-17β-stimulated hydrogen transfer between pyridine nucleotides in placenta occurred by virtue of the reversible oxidation of the steroid by a 17β-hydroxysteroid dehydrogenase in the following manner (173, 182, 183):

Purification of the placental enzyme system and extensive studies of its properties have supported the conclusion that a single 17β-hydroxysteroid dehydrogenase in placenta with dual pyridine nucleotide specificity catalyzes the estrogen-dependent transhydrogenation reaction. The experimental evidence for this contention may be summarized as follows (173, 182, 183): (a) The dehydrogenase and transhydrogenase activities, measured with both the natural pyridine nucleotides and various synthetic analogues, parallel each other during the course of extensive purification.

(b) The steroid specificity of both reactions is identical, since only 17β-hydroxy- or 17-ketosteroids react with both enzyme systems, (c) The pyridine nucleotide and analogue specificity of the two reactions is identical, (d) The competitive interaction between the pyridine nucleotides and their analogues in the dehydrogenase and in the transhydrogenase reactions are closely parallel to each other, (e) During the course of transhydro-genation, estradiol-17β and estrone are interconverted.

The manner in which the hydroxysteroid dehydrogenase which reacts with both DPN and TPN can transfer hydrogen between these pyridine nucleotides may be visualized from the following cyclic formulation, which illustrates hydrogen transfer from TPNH to DPN+, mediated by estradiol-17β:

Since the reactions catalyzed by hydroxysteroid dehydrogenases are reversible under suitable conditions, the above process may equally well be regarded as proceeding from DPNH to TPN+.

The kinetics of the transhydrogenation reactions catalyzed by this soluble placental enzyme are complex. The affinity of the enzyme for the donor and acceptor nucleotides is very greatly different. The strength of binding for TPN and TPNH by the enzyme is probably at least one thousand times higher than for DPN and DPNH. Furthermore, there is a severe competition between pyridine nucleotides when more than one species exists in the reaction mixture. This competition between pyridine nucleotides for a common binding site(s) is clearly evident in both measurements of dehydrogenase and transhydrogenase functions of the placental enzyme. Hydrogen transfer between pyridine nucleotides occurs at very low concentrations of estradiol-17β (< 10^-8 M). The rate and direction of hydrogen transfer in this system is governed by many factors, among which the ratio of concentration of hydrogen donor and acceptor nucleotides and the pH are critical. Because of the great differences in binding constants for the pyridine nucleotides, hydrogen transfer can only occur under highly restricted conditions (182, 183).

Other Hydroxysteroid Dehydrogenases

The soluble fraction of rat liver contains a 3 α-hydroxysteroid dehydrogenase which catalyzes the inter-conversion of a variety of 3 α-hydroxysteroids and 3-ketosteroids, in the presence of either DPN or TPN (Tomkins, G. M. J. Biol. Chem., 218: 437. 1956). Partially purified preparations of this enzyme promote the reversible transfer of hydrogen between TPNH and DPN+ in the presence of very low concentrations of androsterone, androstane-3,17-dione, or other 3 o-hydroxy- and 3-ketosteroids (72, 74). These reactions involve the cyclic oxidation and reduction of the steroid, in the following manner:

Closer examination of the properties of this system revealed a competition between the nucleotides for a common binding site which, like the placental enzyme, has a much higher affinity for TPN and TPNH than for DPN and DPNH. Consequendy, the transfer of hydrogen occurs only with suitable concentrations of donor and acceptor nucleotides. The concentration of androsterone (or androstane-3,17-dione) required for half-maximum rate of hydrogen transfer was about 1 X 10^-6 M (183).

The highly purified 30- and 17β-hydroxysteroid dehydrogenases of P. testosteroni react with DPN and a number of its analogues but are unable to use TPN as a hydrogen acceptor. Consequendy, these bacterial enzymes cannot catalyze hydrogen transfer between DPN and TPN. However, these enzymes promote rapid transfer of hydrogen from DPNH to the 3-acetylpyridine, 3-pyridine aldehyde and the thionicotinamide analogues of DPN in the presence of minute amounts of their steroid substrates (183).

Physiological Implications

Hydroxysteroid dehydrogenases exhibit relatively high efficiency in catalyzing hydrogen transport between pyridine nucleotides in comparison to some other dehydrogenases with dual pyridine nucleotide specificity. It has been suggested that this may be related to the following factors: (i) The equilibria of pyridine nucleotide-linked steroid oxidations favor reversibility near neutral pH, thus assuring the presence of comparable concentrations of steroid alcohol and ketone under such conditions; (ii) the extremely high affinities of the enzymes for certain steroids; and (iii) comparable rates of reaction with both donor and acceptor pyridine nucleotides (182, 183).

Following the discovery that very low concentrations of certain naturally occurring steroid hormones may serve as hydrogen carriers between pyridine nucleotides, it was suggested that this function may constitute, at least in part, the chemical basis of steroid hormone action (74, 173, 182, 183). Several mammalian hydroxysteroid dehydrogenases react with DPN and TPN. These enzymes are for the most part located in the extra-mitochondrial regions of the cell. It was therefore suggested that steroid hormones might exert a controlling influence over the pyridine nucleotide balance in these regions of certain cells. Since the two forms of pyridine nucleotide have the same oxidation-reduction potential, but do not interact freely in the cell, it seemed plausible that a mechanism which could transfer hydrogen between the two forms of pyridine nucleotides might exercise critical metabolic control (182, 183). Two aspects of metabolism in particular were suggested as potential sites of control by steroid-mediated transhydrogenations: (a) energy capture from oxidations of reduced pyridine nucleotides and (6) specific participation of TPNH in biosynthetic reactions. Relatively few experiments designed to assess the physiological role of steroid-mediated hydrogen transport have been carried out. The experimental results available cannot be regarded as conclusively establishing or refuting the regulatory significance of these reactions.