Estrophilic 3 alpha,3 beta,17 beta,20 alpha-hydroxysteroid dehydrogenase from rabbit liver--I. Isolation and purification

A procedure for isolation of a highly-purified estrophilic hydroxysteroid dehydrogenase (EHSD) from rabbit liver, including ammonium sulphate fractionation, gel filtration, ion-exchange and affinity chromatography on estradiol-Sepharose, has been developed. The enzyme possesses NADP-dependent 3 alpha,3 beta,17 beta,20 alpha-HSD activities with a wide spectrum of androgenic, progestagenic, and estrogenic substrates. EHSD is a monomeric protein whose molecular mass determined by different methods is 35,000-39,000. The protein exhibits microheterogeneity due to the differences in molecular surface charge. The catalytic and hormone-binding properties and molecular sizes of the two protein fractions obtained by chromatography on DEAE-Toyopearl are close or identical. The enzymatic activity of EHSD is minor as compared to other HSDs from rabbit liver. However, the low values of Km, the high affinity for steroid ligands, and high tissue levels of EHSD suggest the protein to play a role in the biodynamics of sex hormones.     

Estrophilic 3 alpha,3 beta,17 beta,20 alpha-hydroxysteroid dehydrogenase from rabbit liver--II. Mechanisms of enzyme-steroid interaction

Binding of [3H]estradiol, [3H]testosterone and [3H]progesterone to purified NADP-dependent estrophilic 3 alpha,3 beta,17 beta,20 alpha-hydroxysteroid dehydrogenase (EHSD) from rabbit liver cytosol has been examined. The three steroids bind to the enzyme with moderate [corrected] affinity (Ka congruent to 10(7) [corrected] M-1 at 4 degrees C) and equal binding capacity. High-rates were shown for both association and dissociation processes. The steroids competitively inhibited the binding of each other to EHSD. At the same time, their relative binding affinities (RBA) were dependent on the nature of [3H]ligand. The results of RBA determinations for 72 steroids and their analogues by inhibition of [3H]progesterone binding to EHSD suggest that androgens and gestagens bind preferentially to the same site on EHSD molecule, while estrogens (at least by their D-ring) bind to another site. The assumption that EHSD molecule has more than one binding site for steroids is corroborated by (i) substrate inhibition revealed for a number of steroids; (ii) the estrogen ability to potentiate 20 alpha-reduction of progesterone; (iii) stimulatory effect of 5 alpha (beta)-androstane-3 alpha (beta), 17 beta-diols on [3H]testosterone and progesterone binding; and (iv) reciprocal effect of NADP on [3H]estradiol and [3H]testosterone binding to EHSD. Significant differences in sensitivity to pH and changes in NaCl concentration upon metabolism and binding of various steroids have been found. At concentrations of 16 mM dithiothreitol potentiated catalytic conversion of some steroids and had no effect on metabolism of others. Both the affinity for steroids and binding capacity of EHSD are found to be cofactor-dependent. It is speculated that EHSD has a complex active center including at least two mutually influencing steroid-binding sites tightly related with cofactor-binding site. The polyfunctionality of EHSD may be due to both the excess of functional protein groups that form individual constellations upon binding of any steroid and also to conformational lability of EHSD molecule implying alternative orientations of steroids at the binding site.     

Mechanism-based inactivation of dihydropyrimidine dehydrogenase by 5-ethynyluracil.

Uracil analogues with appropriate substituents at the 5-position inactivated dihydropyrimidine dehydrogenase (DHPDHase). The efficiency of these inactivators was highly dependent on the size of the 5-substituent. For example, 5-ethynyluracil inactivated DHPDHase with an efficiency (kinact/Ki) that was 500-fold greater than that for 5-propynyluracil. 5-Ethynyluracil inactivated DHPDHase by initially forming a reversible complex with a Ki of 1.6 +/- 0.2 microM. This initial complex yielded inactivated enzyme with a rate constant of 20 +/- 2 min-1 (kinact). Thymine competitively decreased the apparent rate constant for inactivation of DHPDHase by 5-ethynyluracil. The absorbance spectrum of 5-ethylnyluracil-inactivated DHPDHase was different from that of reduced enzyme. These optical changes were correlated with the loss of enzymatic activity. 5-Ethynyluracil inactivated DHPDHase with a stoichiometry of 0.9 mol of inactivator per mol of active site. Enzyme inactivated with [2-14C]5-ethynyluracil retained all of the radiolabel after denaturation in 8 M urea, but lost radiolabel under acidic conditions. These results suggested that inactivation was due to covalent modification of an amino acid residue and not due to modification of a noncovalently bound prosthetic group. A radiolabeled peptide was isolated from a tryptic digest of the enzyme inactivated with [2-14C]5-ethynyluracil. The sequence of this peptide was Lys-Ala-Glu-Ala-Ser-Gly-Ala-Y-Ala-Leu-Glu-Leu-Asn-Leu-Ser-X-Pro-His-Gly- Met-Gly-Glu-Arg, where X and Y were unidentified amino acids. Since the radiolabel was lost from the peptide during the first cycle on the amino acid sequenator, the position of the radiolabeled amino acid was not determined. The amino acid residue designated by X was identified as a cysteine from previous work with DHPDHase inactivated with 5-iodouracil. In contrast to 5-ethynyluracil, 5-cyanouracil was a reversible inactivator of the enzyme. 5-Cyanouracil-inactivated enzyme slowly regained activity (t1/2 = 1.8 min) after dilution into the standard assay. DHPDHases isolated from rat, mouse, and human liver had similar sensitivities to inactivation by 5-alkynyluracils.     

Human placental 17 beta-estradiol dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase. Studies with 6 beta-bromoacetoxyprogesterone

Two soluble enzyme activities, 17 beta-estradiol dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase, copurified from the cytosol fraction of human term placenta, were identically inactivated by 6 beta-bromoacetoxyprogesterone. This affinity alkylating steroid binds at the enzyme-active site (Km = 866 microM; Vmax = 0.073 mumol/min/mg). Enzyme inactivation by four concentrations of 6 beta-bromoacetoxyprogesterone (molar ratio of steroid to enzyme, 71/1 to 287/1) causes irreversible and time-dependent loss of both the 17 beta- and 20 alpha-activities according to first order kinetics and affirms that the alkylating steroid is an active site-directed inhibitor (KI = 2.7 X 10(-3) M; k3 = 1.6 X 10(-3) s-1). Affinity radioalkylation studies using 6 beta-[2'-14C]bromoacetoxyprogesterone indicate that 2 mol of steroid are bound to each mole of inactivated enzyme dimer (Mr = 68,000). Amino acid analyses of the acid hydrolysate of radioalkylated enzyme show that 6 beta-bromoacetoxyprogesterone carboxymethylates cysteine (56%), histidine (22%), and lysine (8%) residues in the active site. These results are identical with those reported for 2-bromo[2'-14C]acetamidoestrone methyl ether radioalkylation of purified "17 beta-estradiol dehydrogenase." The parallel inactivation of 17 beta-estradiol dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase by 6 beta-bromoacetoxyprogesterone further shows that both activities reside at a single enzyme-active site. The radioalkylation profile supports our proposed model of one enzyme-active site wherein the bound progestin and estrogen substrates are inverted, one relative to the other.     

The 20 alpha-hydroxysteroid dehydrogenase of Streptomyces hydrogenans

In addition to the well-known 3 alpha,20 beta-hydroxysteroid dehydrogenase ('cortisone reductase'), Streptomyces hydrogenans produces a relatively stable, NAD-dependent 20 alpha-hydroxysteroid dehydrogenase of molecular mass approximately 48 kDa. This enzyme catalyzes the transfer of hydrogen from the 4-pro-S position of NADH.     

Stereospecificity of hydrogen transfer by bovine testicular 20 alpha-hydroxysteroid dehydrogenase

The stereospecificity of hydrogen transfer between steroid (17-hydroxyprogesterone) and both natural cofactors by bovine testicular 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) has been determined. Cofactors used in these studies, [4-pro-S-3H]NADH ([4B-3H]NADH) and [4-pro-S-3H]NADPH ([4B-3H]NADPH) were generated with human placental estradiol 17 beta-dehydrogenase (EC 1.1.1.62) utilizing [17 alpha-3H]estradiol-17 beta and NAD+ or NADP+, respectively. The resulting [4B-3H]NADH and [4B-3H]NADPH were purified by ion-exchange chromatography and separately incubated with molar excess of 17-hydroxyprogesterone as substrate in the presence of 20 alpha-HSD. Following incubation, steroid reactant and product were extracted, separated by HPLC and quantitated as to mass and content of tritium. The oxidized and reduced cofactors were separated by ion-exchange chromatography and quantitated as to mass and tritium content. In all incubations, equimolar amounts of 17,20 alpha-dihydroxy-4-pregnen-3-one and oxidized cofactor were obtained. Further, all recovered radioactivity remained with cofactor and none was found in the steroid product. In additional experiments, both reduced cofactors were separately incubated with glutamate dehydrogenase, an enzyme known to transfer from the B-side of the nicotinamide ring. Here radioactivity was present only in the unreacted cofactor fractions and in the product, glutamic acid. The results indicate that bovine testicular 20 alpha-HSD catalyzes transfer of the 4A-hydrogen from the dihydronicotinamide moiety of the reduced cofactor. Finally, this work described modifications that represent considerable improvement in the purification and assay of bovine 20 alpha-HSD as originally described.     

Kinetic and stereochemical characterization of hamster liver 3 alpha-hydroxysteroid dehydrogenase and 3 alpha(17 beta)-hydroxysteroid dehydrogenase

The kinetic mechanism of NADP(+)-dependent 3 alpha-hydroxysteroid dehydrogenase and NAD(+)-dependent 3 alpha(17 beta)-hydroxysteroid dehydrogenase, purified from hamster liver cytosol, was studied in both directions. For 3 alpha-hydroxysteroid dehydrogenase, the initial velocity and product inhibition studies indicated that the enzyme reaction sequence is ordered with NADP+ binding to the free enzyme and NADPH being the last product to be released. Inhibition patterns by Cibacron blue and hexestrol, and binding studies of coenzyme and substrate are also consistent with an ordered bi bi mechanism. For 3 alpha(17 beta)-hydroxysteroid dehydrogenase, the steady-state kinetic measurements and substrate binding studies suggest a random binding pattern of the substrates and an ordered release of product; NADH is released last. However, the two enzymes transferred the pro-R-hydrogen atom of NAD(P)H to the carbonyl substrate.     

Demonstration of 3 alpha(17 beta)-hydroxysteroid dehydrogenase distinct from 3 alpha-hydroxysteroid dehydrogenase in hamster liver.

NAD(+)-linked and NADP(+)-linked 3 alpha-hydroxysteroid dehydrogenases were purified to homogeneity from hamster liver cytosol. The two monomeric enzymes, although having similar molecular masses of 38,000, differed from each other in pI values, activation energy and heat stability. The two proteins also gave different fragmentation patterns by gel electrophoresis after digestion with protease. The NADP(+)-linked enzyme catalysed the oxidoreduction of various 3 alpha-hydroxysteroids, whereas the NAD(+)-linked enzyme oxidized the 3 alpha-hydroxy group of pregnanes and some bile acids, and the 17 beta-hydroxy group of testosterone and androstanes. The thermal stabilities of the 3 alpha- and 17 beta-hydroxysteroid dehydrogenase activities of the NAD(+)-linked enzyme were identical, and the two enzyme activities were inhibited by mixing 17 beta- and 3 alpha-hydroxysteroid substrates, respectively. Medroxyprogesterone acetate, hexoestrol and 3 beta-hydroxysteroids competitively inhibited 3 alpha- and 17 beta-hydroxysteroid dehydrogenase activities of the enzyme. These results show that hamster liver contains a 3 alpha(17 beta)-hydroxysteroid dehydrogenase structurally and functionally distinct from 3 alpha-hydroxysteroid dehydrogenase.     

Cofactor requirements of steroid-17-20-desmolase and 20 alpha-hydroxysteroid dehydrogenase activities in cell extracts of Clostridium scindens

Two neutral steroid-transforming activities were demonstrated in cell extracts of Clostridium scindens. Steroid-17-20-desmolase and 20 alpha-hydroxysteroid dehydrogenase were found to be inducible in cells cultured in the presence of cortisol. Both activities required manganese ions and NAD+ or NADH for activity. Cortisol, cortisone and 11-desoxycortisol were substrates as well as inducers of steroid-17-20-desmolase and 20 alpha-hydroxysteroid dehydrogenase activities. 17 alpha-Hydroxyprogesterone was an effective inducer but did not serve as a substrate for either enzyme activity. C. scindens is the first bacterial species of the normal human intestinal flora reported to elaborate inducible steroid-17-20-desmolase and 20 alpha-hydroxysteroid dehydrogenase activities. The results of cofactor, substrate specificity and induction studies suggest that these two activities may reside in the same enzyme complex.     

Purification and characterization of rat ovarian 20 alpha-hydroxysteroid dehydrogenase

To investigate the regulatory mechanism of 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) (EC 1.1.1.149) activity in ovarian tissue, the enzyme was purified from ovaries of normal mature female rats. Column chromatography of the cytosolic fraction from ovaries on DEAE-Toyopearl 650M revealed two peaks of the 20 alpha-HSD activity at different ionic strengths. These peaks were designated HSD1 and HSD2, respectively. Each of the active fractions was further purified to homogeneity by dye-affinity chromatography using Matrex Green A and AF Red-Toyopearl. Both the fractions appeared as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (at Mr = 33,000 under reducing conditions). Under non-reducing conditions, similar values were obtained on gel-exclusion HPLC, indicating that the enzyme fractions were single-stranded, monomeric polypeptides. Homogeneous HSD1 and HSD2 were purified 361-fold and 509-fold, respectively, and differed in their substrate preference. The two enzyme fractions had Km values of 4.75 microM and 5.16 microM for 20 alpha-dihydroprogesterone, respectively, and showed almost the same RF values on reverse-phase HPLC and free-zone capillary electrophoresis. However, amino acid composition was slightly different, i.e. lysin content was higher in HSD1 than HSD2. Thus, it was clarified that two types of 20 alpha-HSD with very similar molecular structures are present in the rat ovary.     

Oxidative 3alpha-hydroxysteroid dehydrogenase activity of human type 10 17beta-hydroxysteroid dehydrogenase

In vitro enzyme assays have demonstrated that human type 10 17beta-hydroxysteroid dehydrogenase (17beta-HSD10) catalyzes the oxidation of 5alpha-androstane-3alpha,17beta-diol (adiol), an almost inactive androgen, to dihydrotestosterone (DHT) rather than androsterone or androstanedione. To further investigate the role of this steroid-metabolizing enzyme in intact cells, we produced stable transfectants expressing 17beta-HSD10 or its catalytically inactive Y168F mutant in human embryonic kidney (HEK) 293 cells. It was found that DHT levels in HEK 293 cells expressing 17beta-HSD10, but not its catalytically inactive mutant, will dramatically increase if adiol is added to culture media. Moreover, certain malignant prostatic epithelial cells have more 17beta-HSD10 than normal controls, and can generate DHT, the most potent androgen, from adiol. This event might promote prostate cancer growth. Analysis of the 17beta-HSD10 sequence shows that this enzyme does not have any ER retention signal or transmembrane segments and has not originated by divergence from a retinol dehydrogenase. The data suggest that the unique mitochondrial location of this HSD [Eur. J. Biochem. 268 (2001) 4899] does not prevent it from oxidizing the 3alpha-hydroxyl group of a C19 sterol in living cells. The experimental results lead to the conclusion that mitochondrial 17beta-HSD10 plays a significant part in a non-classical androgen synthesis pathway along with microsomal retinol dehydrogenases.     

20alpha-hydroxysteroid dehydrogenase: a T lymphocyte-associated enzyme.

20alpha-Hydroxysteroid dehydrogenase (20alpha-SDH), an enzyme which reduces progesterone to 20alpha-dihydroprogesterone, was found to be associated with T lymphocytes. 20alphaSDH activity was present in spleen cells bearing theta antigen, spleen cells nonadherent to nylon wool (T lymphocyte-enriched population), and in thymocytes. Almost no enzymatic activity was found in bone marrow cells from normal mice and in spleen cells from neonatally thymectomized or athymic nude mice. T cell mitogens (PHA and Con A), but not the B cell mitogen LPS, induced high levels of enzymatic activity 48 hr after addition to spleen cell cultures. The level of 20alphaSDH activity in lymphocytes was age dependent. At the age of 4 weeks 20alphaSDH activity in thymocytes, spleen cells, and lymph node lymphocytes was 3 to 5 times higher than at 8 and 16 weeks. Progesterone (5.0 X 10(-7) M) was found to inhibit thymocyte proliferation after exposure to mitogens, but not 20alpha-dihydroprogesterone (10(-6) M). 20alpha SDH may protect the embryonic thymocytes against high concentrations of progesterone.     

3(20)alpha-hydroxysteroid dehydrogenase activity of monkey liver indanol dehydrogenase

Homogeneous indanol dehydrogenase from monkey liver catalyzed the reversible conversion of 3 alpha- or 20 alpha-hydroxy groups of several bile acids and 5 beta-pregnanes to the corresponding 3- or 20-ketosteroids. The kcat values for the steroids determined at pH 7.4 were low, but the kcat/Km values for the 3-ketosteroids were comparable to or exceeded those for 1-indanol and xenobiotic carbonyl substrates. The enzyme transferred the 4-pro-R-hydrogen atom of NADPH to the 3 beta- or 20 beta-face of the ketosteroid substrate. Competitive inhibition of the hydroxysteroid dehydrogenase activity of the enzyme by medroxyprogesterone acetate, hexestrol, and 1,10-phenanthroline suggests that both 1-indanol and hydroxysteroid are oxidized at the same active site on the enzyme. The specific inhibitor of the enzyme, 1,10-phenanthroline, suppressed the 3 alpha-hydroxysteroid dehydrogenase activity in the crude extract of monkey liver by 50%. The results strongly suggest that indanol dehydrogenase acts as a 3(20)alpha-hydroxysteroid dehydrogenase in the metabolism of certain steroid hormones and bile acids.     

Mechanism-based inactivation of human glutaryl-CoA dehydrogenase by 2-pentynoyl-CoA: rationale for enhanced reactivity

2-Pentynoyl-CoA inactivates glutaryl-CoA dehydrogenase at a rate that considerably exceeds the rates of inactivation of short chain and medium chain acyl-CoA dehydrogenases by this inhibitor and related 2-alkynoyl-CoAs. To determine the rate of inactivation by 2-pentynoyl-CoA, we investigated the inactivation in the presence of a non-oxidizable analog, 3-thiaglutaryl-CoA, which competes for the binding site. The enhanced rate of inactivation does not reflect an alteration in specificity for the acyl group, nor does it reflect the covalent modification of a residue other than the active site glutamate. In addition to determining the inactivation of catalytic activity a spectral intermediate was detected by stopped-flow spectrophotometry, and the rate constants of formation and decay of this charge transfer complex (lambdamax approximately 790 nm) were determined by global analysis. Although the rate-limiting step in the inactivation of the other acyl-CoA dehydrogenases can involve the abstraction of a proton at C-4, this is not the case with glutaryl-CoA dehydrogenase. Glutaryl-CoA dehydrogenase is also differentiated from other acyl-CoA dehydrogenases in that the catalytic base must access both C-2 and C-4 in the normal catalytic pathway. Access to C-4 is not obligatory for the other dehydrogenases. Analysis of the distance from the closest carboxylate oxygen of the glutamate base catalyst to C-4 of a bound acyl-CoA ligand for medium chain, short chain, and isovaleryl-CoA dehydrogenases suggests that the increased rate of inactivation reflects the carboxylate oxygen to ligand C-4 distance in the binary complexes. This distance for wild type glutaryl-CoA dehydrogenase is not known. Comparison of the rate constants of inactivation and formation of a spectral species between wild type glutaryl-CoA dehydrogenase and a E370D mutant are consistent with the idea that this distance in glutaryl-CoA dehydrogenase contributes to the enhanced rate of inactivation and the 1,3-prototropic shift catalyzed by the enzyme.     

Isolation of 3 beta,20 alpha-hydroxysteroid oxidoreductase from sheep fetal blood

3 beta,20 alpha-Hydroxysteroid oxidoreductase has been isolated from ovine fetal blood by a 2,370-fold purification scheme of ammonium sulfate fractionation, calcium phosphate gel adsorption, affinity chromatography, and fast performance liquid chromatography. A new high performance liquid chromatography-based assay for measuring 20 alpha-reductase activity is described. The enzyme is a monomer with a molecular weight of 35,000 and uses NADPH as a cofactor for reductase activity. It reduces progesterone to 4-pregnen-20 alpha-ol-3-one or 5 alpha-dihydrotestosterone to 5 alpha-androstan-3 beta,17 beta-diol with kinetic characteristics of Km = 30.8 microM and Vmax = 0.7 nmol min-1 (nmol of enzyme)-1 or Km = 74 microM and Vmax = 1.3 nmol min-1 (nmol of enzyme)-1, respectively. 5 alpha-Dihydrotestosterone competitively inhibits 20 alpha-reductase activity with a Ki value of 102 microM.     

Mechanism-based inactivation of dopamine beta-monooxygenase by beta-chlorophenethylamine

Functionalization of the beta-carbon of phenethylamines has been shown to produce a new class of substrate/inhibitor of dopamine beta-monooxygenase. Whereas both beta-hydroxy- and beta- chlorophenethylamine are converted to alpha-aminoacetophenone at comparable rates, only the latter conversion is accompanied by concomitant enzyme inactivation ( Klinman , J. P., and Krueger , M. (1982) Biochemistry 21, 67-75). In the present study, the nature of the reactive intermediates leading to dopamine beta-monooxygenase inactivation by beta- chlorophenethylamine has been investigated employing kinetic deuterium isotope effects and oxygen- 18 labeling as tools. Mechanistically significant findings presented herein include: 1) an analysis of primary deuterium isotope effects on turnover, indicating major differences in rate-determining steps for beta-chloro- and beta- hydroxyphenethylamine hydroxylation, Dkcat = 6.1 and 1.0, respectively; 2) evidence that dehydration of the gem-diol derived from oxygen- 18-labeled beta- hydroxyphenethylamine hydroxylation occurs in a random manner, attributed to dissociation of enzyme-bound gem-diol prior to alpha-aminoacetophenone formation; 3) the observation of a deuterium isotope effect for beta- chlorophenethylamine inactivation, Dkinact = 3.7, implicating C--H bond cleavage in the inactivation process; and 4) the demonstration that alpha-aminoacetophenone can replace ascorbic acid as exogenous reductant in the hydroxylation of tyramine. As discussed, these findings support the intermediacy of enzyme-bound alpha-aminoacetophenone in beta- chlorophenethylamine inactivation, and lead us to propose an intramolecular redox reaction to generate a ketone-derived radical cation as the dopamine beta-monooxygenase-inactivating species.     

Estrogen induction of 20alpha-hydroxysteroid dehydrogenase in mouse vaginal tissue

The conversion of progesterone to 20α-hydroxy-4-pregnen-3-one by 20α-hydroxysteroid dehydrogenase was measured in mouse vaginal tissue. The enzyme was confined to the 105,000 × g supernatant of tissue homogenates and the requirement for reduced NADP demonstrated. The Initial rates of 20α-hydroxysteroid dehydrogenase were determined in the cytosol of tissues from four-day estrogen-treated and untreated animals. The rate of 20α-hydroxy-4-pregnen-3-one formation per vagina was increased 15-fold by estrogen stimulation. This increase could not be accounted for on the basis of increased organ weight or increased availability of cofactor. These findings indicate that 20α-hydroxy steroid dehydrogenase induction in the mouse vaginae is under estrogen control.