A 17 β-hydroxysteroid dehydrogenase has been obtained as an adaptive enzyme from Pseudotnonas testosteroni and purified (83) to a considerable extent (activity = 30-50 µmoles DPN reduced per minute per milligram protein at 25°C). This enzyme reacts with neutral and phenolic steroids of the C18 and C19 series and requires DPN or certain of its analogs, but does not react with TPN. The substrate binding and specificity of this enzyme have been examined in some detail (82, 110). The Michaelis constant for estradiol-17β is very low (< 10-7M) whereas that for testosterone is about 1 X 10-6M. The same enzyme apparently catalyzes also a DPN-dependent oxidation of various 3P-hydroxysteroids.

A soluble 17β-hydroxysteroid dehydrogenase from human placenta has been partially purified by Langer and Engel (73, 74). This enzyme has a high degree of specificity for 17β-hydroxysteroids in which ring A is aromatic; testosterone and 19-nortestosterone were oxidized at less than 5% the rate of estradiol-17β\ The activities for aromatic and neutral 17β-hydroxysteroids followed each other during purification, but conceivably may reside in different proteins. This placental enzyme was purified in our laboratory (112). The most active preparations catalyzed the reduction of 0.2 umole DPN per minute per milligram protein at 25°C, and reacted with DPN and TPN at comparable rates. Recently, Yamasaki and colleagues (143) have fractionated from rat liver a 3β-hydroxysterol dehydrogenase which oxidizes cholesterol to 4-cholestenone, but does not attack 3β-hydroxy-steroids lacking a side chain. This enzyme reacts equally well with DPN and TPN. The same preparations also oxidize testosterone to 4-androstene-3,17-dione, and may be contaminated with the DPN-linked 17β-hydroxy-steroid dehydrogenase activity first reported by Sweat and associates in steer livers (108). The reactivity of the latter enzyme with TPN is not reported. Liver microsomes contain an 11β-hydroxysteroid dehydrogenase which interconverts Cortisol and cortisone and has a dual pyridine nucleotide specificity (56).

Certain other interconversions of hydroxy- and ketosteroids have not been adequately characterized but probably require the participation of pyridine nucleotides. These are: the oxidation of 36-hydroxysteroids by various endocrine tissues (101) and especially by adrenal microsomes (14); the reduction of 20-ketosteroids to 20a- and 20β-hydroxysteroids by liver microsomes (24) and by ovarian homogenates (136). In a preliminary communication, Kochakian and co-workers (29) have reported that guinea pig liver and kidney contain a mitochondrial DPN-linked 17β-hydroxy-steroid dehydrogenase for testosterone, as well as a second enzyme of similar steroid specificity which reacts with TPN and is principally associated with the "supernatant" fraction. In such crude systems, it is probably not justified to assign precise pyridine nucleotide specificities because of the obvious problems of interference by contaminating enzymes which may actively degrade or oxidize DPN(H) and TPN(H) at different rates. It is, nevertheless, striking that the four mammalian hydroxysteroid dehydrogenases which have been purified sufficiently for direct spectrophotometric study, all react with both DPN and TPN at comparable rates.

Detailed investigations of the binding of steroids to purified hydroxysteroid dehydrogenases have revealed that the affinities are high in comparison with most enzyme-substrate complexes (110). It has also been shown that the binding process is highly specific and sometimes extraordinarily sensitive to even relatively minor alterations in the geometry or sub-stituents of the steroid structure. The interaction of the enzymes with their steroidal substrates appears to involve many points of attachment other than the polar oxygenated groups, and seems to extend over most of the steroid skeleton (110).

Hydroxysteroid dehydrogenases, like other pyridine nucleotide-linked enzymes (78, 123), catalyze a direct transfer of hydrogen which is stereospecific for substrate and pyridine nucleotide. With purified bacterial 3a-, (3- and 17-)β-hydroxysteroid dehydrogenases as well as purified placental 176-hydroxysteroid dehydrogenase (with both DPN and TPN) the hydrogen transfer has been shown to involve side II (p) of the pyridine ring (59, 113, 114).

Hydroxysteroid dehydrogenases require free sulfhydryl groups for activity and are readily inactivated by heavy metals, but may be stabilized against denaturation by certain steroids or pyridine nucleotides . The reactions they catalyze are freely reversible. The equilibrium constants, K = [steroid ketone] [DPNH] [H+]/[steroid alcohol] [DPN+], have been measured for a variety of compounds with the aid of highly purified a- and β-hydroxy-steroid dehydrogenases derived from Pseudotnonas testosteroni. The equilibrium constants lie in the range of (1 to 40) X 108M at 25°C (110, 113). The magnitudes of the individual values are, in part, dependent upon the conformations of the groups undergoing oxidation. Since these pyridine nucleotide-linked oxidations involve the stoichiometric participation of a proton, their equilibria are strictly pH-dependent. The equilibrium constants of these reactions are such as to favor the existence of significant amounts of both hydroxy- and ketosteroids at physiological hydrogen ion concentrations. Thus, assuming equimolar concentrations of the oxidized and reduced forms of pyridine nucleotides, it may be shown that at equilibrium at pH 7.0 the ratios of concentrations of steroid ketones to alcohols lie in the range of 0.01 to 0.4 for various compounds with oxygenated functions at C-3 and C-17.

III. The Identity And Properties Of The Estrogen-Mediated Placental Transhydrogenase

Villee and co-workers (36, 37, 125, 128) discovered that upon the direct addition of minute amounts of estradiol-17β or estrone to slices of human placenta or endometrium, the rates of oxidation of a variety of substrates were enhanced. Upon further examination of this effect with soluble extracts of human placenta, these authors (33, 127) showed a stimulatory effect of estrogenic hormones upon the reduction of DPN (but not of TPN) in the presence of isocitrate. These findings were interpreted as showing that the hormone activated (in an unknown manner) an inactive form of a DPN-linked isocitric dehydrogenase. Although active TPN-specific isocitric dehydrogenases occur in the soluble cell sap and particulate fractions of many tissues, a DPN-specific isocitric dehydrogenase of animal tissues had been described previously only in the mitochondria (96). Experiments in our laboratory showed that the estrogen-sensitive placental enzyme system required catalytic quantities of TPN, and was in fact a pyridine nucleotide transhydrogenating system which was in no special way related to the oxidation of isocitrate (117). The isocitrate in conjunction with a soluble TPN-specific dehydrogenase merely served to generate TPNH and could be replaced by TPNH, added as such, or by other TPN-reducing systems. This led to the formulation of the following coupled reactions to account for the estrogen effect:

Our findings were quickly confirmed by both Villee (129) and by Hollander (48). Villee (129) claimed that the transhydrogenase reaction did not proceed in the reverse direction (i.e, from DPNH to TPN), but we have demonstrated that the reaction is readily reversible under appropriate conditions (112).

Previous to this finding of a soluble mammalian pyridine nucleotide transhydrogenating system, enzymes of this type had been found only in the intracellular particles of some animal tissues (64). Independent studies by Langer and Engel (73, 74) on the metabolism of estrogens by placenta, had revealed the presence of a soluble pyridine nucleotide-dependent 176-hydroxysteroid dehydrogenase which was purified and partially characterized. This enzyme was found to possess high affinity for estradiol-17β and estrone. It promoted reactions which were readily reversible, and reacted almost equally well with both DPN and TPN. Our investigations (112, 117) of the mechanism and properties of the soluble placental transhydrogenating system have pointed to its identity with the 178-hydroxysteroid dehydrogenase, reacting as follows: