Synthesis of a steroidal A ring aromatic sulfonic acid as an inhibitor of steroid 5 alpha-reductase

The synthesis of 17 beta-(N,N-diisopropylcarbamoyl)estra-1,3,5(10)-triene-3-sulfonic acid (3) has been accomplished. Sulfonate 3 was designed as a novel inhibitor of human steroid 5 alpha-reductase based on considerations of enzyme mechanisms, and exhibits an inhibition constant in the low nanomolar range.     

Inactivation of human placental 17 beta,20 alpha-hydroxysteroid dehydrogenase by 16-methylene estrone, an affinity alkylator enzymatically generated from 16-methylene estradiol-17 beta

The substrate 16-methylene estra-1,3,5(10)-triene-3,17 beta-diol (16-methylene estradiol-17 beta) and its enzyme-generated alkylating product, 3-hydroxy-16-methylene estra-1,3,5(10)-triene-17-one (16-methylene estrone), were synthesized to study the 17 beta- and 20 alpha-hydroxysteroid dehydrogenase activities which coexist in homogeneous enzyme purified from human placental cytosol. 16-Methylene estradiol, an excellent substrate (Km = 8.0 microM; Vmax = 2.8 mumol/mg/min) when enzymatically oxidized to 16-methylene estrone in the presence of NAD+ (256 microM), inactivates simultaneously the 17 beta- and 20 alpha-activities in a time-dependent and irreversible manner following pseudo-first order kinetics (t1/2 = 1.0 h, 100 microM, pH 9.2). 16-Methylene estradiol does not inactivate the enzyme in the absence of NAD+. 16-Methylene estrone (Km = 2.7 microM; Vmax = 2.9 mumol/mg/min) is an affinity alkylator (biomolecular rate constant k'3 = 63.3 liters/mol-s, pH 9.2; KI = 261 microM; k3 = 8.0 X 10(-4) S-1, pH 7.0) which also simultaneously inhibits both activities in an irreversible time-dependent manner (at 25 microM; t1/2 = 7.2 min, pH 9.2; t1/2 = 2.7 h, pH 7.0). Substrates (estradiol-17 beta, estrone, and progesterone) protect against inhibition of enzyme activity by 16-methylene estrone and 16-methylene estradiol. Affinity radioalkylation studies using 16-methylene [6,7-3H]estrone demonstrate that 1 mol of alkylator binds per mol of inactivated enzyme dimer. Thus, 16-methylene estradiol functions as a unique substrate for the enzymatic generation of a powerful affinity alkylator of 17 beta,20 alpha-hydroxysteroid dehydrogenase and should be a useful pharmacological tool.     

C20 steroids: The metabolism of 3-hydroxy-19-nor[21-14C]pregna-1,3,5(10)-trien-20-one in the rabbit

1. The metabolism of 3-hydroxy-19-norpregna-1,3,5(10)-trien-20-one, a possible product of the aromatization of progesterone or pregnenolone, has been studied. 2. After oral administration of this C(20) steroid as the 21-(14)C-labelled compound to two groups of rabbits, the excretion pattern of metabolites in the urine was examined. 3. At 14 days after administration, 3.3-6.5% of the radioactivity had appeared in the urine, 71-79% in the faeces and approx. 10% remained in the gut. 4. A metabolite, isolated from urine mainly as the unconjugated steroid, was identified as 19-norpregna-1,3,5(10)-triene-3,20alpha-diol and constituted 18.5-22% of the total urinary radioactivity. 5. A minor component of the urinary unconjugated steroids was identified as 19-norpregna-1,3,5(10)-triene-3,17alpha,20alpha-triol. 6. A further 2-7.5% of the total urinary radioactivity, isolated only from the urinary sulphate fraction, was tentatively identified as an 18-oxygenated derivative of the administered steroid.     

Inactivation of delta 5-3-oxo steroid isomerase with active-site-directed acetylenic steroids.

Several steroid analogues containing conjugated acetylenic ketone groups as part of a seco-ring structure or as substituents on the intact steroid system are irreversible inhibitors of delta 5-3-oxo steroid isomerase (EC 5.3.3.1) from Pseudomonas testosteroni. Thus 10 beta-(1-oxoprop-2-ynyl)oestr-4-ene-3,17-dione (I), 5,10-seco-oestr-4-yne-3,10,17-trione (II), 17 beta-hydroxy-5,10-seco-oestr-4-yne-3,10-dione (III) and 17 beta-(1-oxoprop-2-ynyl)androst-4-en-3-one (IV) irreversibly inactivate isomerase in a time-dependent manner. In all cases saturation kinetics are observed. Protection against inactivation is afforded by the powerful competitive inhibitor 19-nortestosterone. The inhibition constants (Ki) for 19-nortestosterone obtained from such experiments are in good agreement with those determined from conventional competitive-inhibition studies of enzyme activity. These compounds thus appear to be active-site directed. In every case the inactivated enzyme could be dialysed without return of activity, indicating that a stable covalent bond probably had formed between the steroid and enzyme. Compound (I) is a very potent inhibitor of isomerase [Ki = 66.0 microM and k+2 = 12.5 x 10(-3) s-1 (where Ki is the dissociation constant of the reversible enzyme-inhibitor complex and k+2 is the rate constant for the inactivation reaction of the enzyme-inhibitor complex)] giving half-lives of inactivation of 30-45 s at saturation. It is argued that the basic-amino-acid residue that abstracts the intramolecularly transferred 4 beta-proton in the reaction mechanism could form a Michael-addition product with compound (I). In contrast, although compound (IV) has a lower inhibition constant (Ki = 14.5 microM), it is a relatively poor alkylating agent (k+2 = 0.13 x 10(-3) s-1). If the conjugated acetylenic ketone groups are replaced by alpha-hydroxyacetylene groups, the resultant analogues of steroids (I)-(IV) are reversible competitive inhibitors with Ki values in the range 27-350 microM. The enzyme binds steroids in the C19 series with functionalized acetylenic substituents at C-17 in preference to steroids in the C18 series bearing similar groups in the ring structure or as C-10 substituents. In the 5,10-seco-steroid series the presence of hydroxy groups at both C-3 and C-17 is deleterious to binding by the enzyme.     

Mechanism of inactivation of 3-oxosteroid delta 5-isomerase by 17 beta-oxiranes

The affinity label (17S)-spiro[estra-1,3,5(10),6,8-pentaene-17,2'-oxiran]-3-ol (5 beta) inactivates 3-oxosteroid delta 5-isomerase from Pseudomonas testosteroni by formation of a covalent bond between Asp-38 of the enzyme and the steroid. High-performance liquid chromatography (HPLC) analysis of tryptic digests of inactivated enzyme shows that two isomeric steroid-containing peptides are formed in a ratio of 9:1 at pH 7 (TPS1 and TPS2). Hydrolysis of each of these peptides produces a different steroid: TPS1 releases 17 alpha-(hydroxymethyl)estra-1,3,5(10),6,8-pentaene-3,17 beta-diol (S1) whereas TPS2 yields 17 beta-(hydroxymethyl)estra-1,3,5(10),6,8-pentaene-3,17 alpha-diol (S2). Inactivation of the enzyme by (17S)-spiro[estra-1,3,5(10),6,8-pentaene-17,2'-oxiran-18O]-3-ol, followed by mass spectral analysis of the diacetate of the steroid released upon hydrolysis of the enzyme-inhibitor bond, reveals that TPS1 is formed by attack of Asp-38 at the methylene carbon of the oxirane. In contrast, TPS2 is produced by Asp-38 attack at the tertiary carbon. These results imply that inactivation occurs through concurrent SN1 and SN2 reactions of Asp-38 with the protonated inhibitor and that Asp-38 is located on the alpha face of the steroid when it is bound to the active site in the correct manner to react for both the SN1 and SN2 processes.     

Neighboring group participation. Part 16. Stereoselective synthesis and receptor-binding examination of the four stereoisomers of 16-bromomethyl-3,17-estradiols

The four possible isomers of 3-benzyloxy-16-hydroxymethylestra-1,3,5(10)-trien-17-ol (1a-4a) with proven configurations were converted into the corresponding 3-benzyloxy-16-bromomethylestra-1,3,5(10)-triene-3,17-diols (5e-8e). Depending on the reaction conditions the cis isomers of 3-benzyloxy-16-hydroxymethylestra-1,3,5(10)-trien-17-ol (1a and 2a) were transformed into 3-benzyloxy-16-bromomethylestra-1,3,5(10)-trien-17-yl acetate (5b and 6b) or 16-bromomethyl-3-hydroxyestra-1,3,5(10)-trien-17-yl acetate (5c and 6c) on treatment with HBr and acetic acid. The mechanism of the process can be interpreted as involving front-side neighboring group participation. Under similar experimental conditions, the trans isomers (3a and 4a) yielded only 3-benzyloxy-16-acetoxymethylestra-1,3,5(10)-trien-17-yl acetates (3b and 4b) or 16-acetoxymethylestra-1,3,5(10)-triene-3,17-diyl diacetates (3d and 4d). Both the cis (1a and 2a) and the trans (3a, and 4a) isomers were transformed into 16-bromomethylestra-1,3,5(10)-trien-17-ol (5a-8a) by the Appel reaction on treatment with CBr4/Ph3P. Debenzylation of 5a-8a was carried out with HBr and acetic acid to yield 5e-8e. The debenzylation process in the presence of acetic anhydride produces the diacetates 5d-8d. The structures of the compounds were determined by means of MS, 1H NMR and 13C NMR spectroscopic methods. Compounds 5c-8c and 5e-8e were tested in a radioligand-binding assay. Except for the affinity of 7e for the estrogen receptor (Ki=2.55 nM), the affinities of the eight compounds (5c-8c and 5e-8e) for the estrogen, androgen and progesterone receptors are low (Ki > 0.55, 0.52 and 0.21 microM, respectively).     

Species differences in biotransformation of 17α-cyanomethylestradiol 3-methyl ether

Following oral administration of 9,11- ³H-17α-cyano-methylestra-1,3,5(10)-triene-3,17-diol 3-methyl ether, urinary metabolites were studied in man, baboon, beagle dog, minipig and rat. The metabolite pattern revealed remarkable species differences, especially in quantitative respects. 17α-Cyanomethylestra-1,3,5(10)-triene-3,17-diol, 17α-cyanomethylestra-1,3,5(10)-triene-2,3,17-triol 2-methyl ether, 17α-hydroxymethylestra-1,3,5(10)-triene-3,17-diol and 17α-cyanomethylestra-1,3,5(10)-triene-3,16$\text{6}\text{5}$,17-triol were isolated as principal metabolites. In rat bile, a metabolite was tentatively identified as aγ-lactone of a 17α-carbozymethyl-16α-hydroxy compound.     

Binding, X-ray and NMR studies of the three A-ring isomers of natural estradiol

The effect of the position of the phenolic hydroxyl on the conformations of the three A-ring isomers of estradiol, namely, estra-1,3,5(10)-trien-1,17 beta-diol (10), estra-1,3,5(10)-trien-2,17 beta-diol (3), and estra-1,3,5(10)-trien-4,17 beta-diol (6), has been analyzed by X-ray crystallography. The results of these analyses were correlated with the absorptions of the angular methyl groups in the [1H]NMR spectra of these isomers and natural estradiol (E2). It was observed that the changes in chemical shift of protons at C18 corresponded to skeletal modifications in the steroid structure which changed the anisotropic effect of the hydroxyl group at C17. Examination of the affinity of these A-ring isomers of E2 for the estrogen receptor has shown the 2-hydroxylated isomer 3 to retain 1/5th the affinity of E2 for its binding protein. The 1- and 4-hydroxylated derivatives (10 and 6, respectively) bound to a much lesser extent. The receptor affinities of these estrogen analogues may be related to the angle between the 18-methyl and the 17 beta-hydroxyl groups (or the dihedral angle between the planar A-ring and the angular C18 methyl) as well as the position of the A-ring hydroxyl group.     

Synthesis,structure, and biological properties of some 8α analogues of steroid estrogens with fluorine in position 2

A total synthesis of 8α analogues of steroid estrogens with fluorine in position 2 was achieved. Structural features of these compounds were studied by the example of 17β-acetoxy-2-fluoro-3-methoxy-8α-estra-1,3,5(10)-triene. It was shown that the 8α analogues of 2-fluorosubstituted steroid estrogens have a low uterotropic activity and retain the osteoprotective and hypocholesterolemic activities.     

The genes encoding the hydroxylase of 3-hydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione in steroid degradation in Comamonas testosteroni TA441

Steroid degradation genes of Comamonas testosteroni TA441 are encoded in at least two gene clusters: one containing the meta-cleavage enzyme gene tesB; and another consisting of ORF18, 17, tesI, H, ORF11, 12, and tesDEFG. TesH and I are, respectively, the Delta(1)- and Delta(4)(5alpha)-dehydrogenase of the 3-ketosteroid, TesD is the hydrolase for the product of meta-cleavage reaction, and TesEFG degrade one of the product of TesD. In this report, we describe the identification of the function of ORF11 (tesA2) and 12 (tesA1). The TesA1- and TesA2-disrupted mutant accumulated two characteristic intermediate compounds, which were identified as 3-hydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione (3-HSA) and its hydroxylated derivative, 3,17-dihydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione by MS and NMR analysis. A complementation experiment using a broad-host range plasmid showed that both TesA1 and A2 are necessary for hydroxylation of 3-HSA to 3,4-dihydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione (3,4-DHSA).     

Synthesis and biologic activities of 11 beta-substituted estradiol as potential antiestrogens

The effect of attachment of a dimethylaminoethoxy or a dimethylaminopropoxy group at the 11 beta-position of estradiol (E2) on its relative binding affinity (RBA) to estrogen receptor (ER) and intrinsic biologic activity is described. The binding of 11 beta-[2-(N,N-dimethylamino) ethoxy]estra-1,3,5(10)-triene-3,17 beta-diol (4) and 11 beta-[3-(N,N- dimethylamino)propoxy]estra-1,3,5(10)-triene-3,17 beta-diol (5) to the ER from immature rat uterine tissue was measured relative to that of [3H]E2 by a competitive binding assay. It was found that the 11 beta-substituted E2 analogs have considerably lower RBA to ER than the corresponding parent compound. The intrinsic activity of compounds 4 and 5 were studied in terms of uterotrophic and antiuterotrophic activity. It was found that the uterotrophic activity of these compounds was drastically reduced compared with E2. However, no antiuterotrophic activity was observed in these compounds at dosages ranging from 1 to 100 micrograms/rat/d.     

Steroid catechol degradation: disecoandrostane intermediates accumulated by Pseudomonas transposon mutant strains

Eleven transposon mutant strains affected in bile acid catabolism were each found to form yellow, muconic-like intermediates from bile acids. To characterize these unstable intermediates, media from the growth of one of these mutants with deoxycholic acid was treated with ammonia, then the crude product was methylated with diazomethane. Four compounds were subsequently isolated; spectral evidence suggested that they were methyl 12 alpha-hydroxy-3-oxo-23,24-dinorchola-1,4-dien-22-oate, methyl 4-aza-12 beta-hydroxy-9(10)-secoandrosta-1,3,5-triene-9,17-dione-3-carboxyl ate, 4-aza-9 alpha, 12 beta-dihydroxy-9(10)-secoandrosta-1,3,5-trien-17-one-3- methyl carboxylate and 4 alpha-[3'-propionic acid]-5-amino-7 beta-hydroxy-7 alpha beta-methyl- 3a alpha, 4,7,7a-tetrahydro-1-indanone-delta-lactam. It is proposed that the mutants are blocked in the utilization of such muconic-like compounds as the 3,12 beta-dihydroxy-5,9,17-trioxo-4(5),9(10)- disecoandrostal (10),2-dien-4-oic acid formed from deoxycholic acid. A further mutant was examined, which converted deoxycholic acid to 12 alpha-hydroxyandrosta-1,4-dien-3,17-dione, but accumulated yellow products from steroids which lacked a 12 alpha-hydroxy function, such as chenodeoxycholic acid. The products from the latter acid were treated as above; spectral evidence suggested that the two compounds isolated were methyl 4-aza-7-hydroxy-9(10)-secoandrosta-1,3,5- triene-9,17-dione-3-carboxylate and 4 alpha-[1'alpha-hydroxy-3'-propionic acid]-5-amino-7a beta-methyl-3a alpha,4,7,7a-tetrahydro-1-indanone-delta-lactam.     

Steroid degradation in Comamonas testosteroni

Steroid degradation by Comamonas testosteroni and Nocardia restrictus have been intensively studied for the purpose of obtaining materials for steroid drug synthesis. C. testosteroni degrades side chains and converts single/double bonds of certain steroid compounds to produce androsta-1,4-diene 3,17-dione or the derivative. Following 9α-hydroxylation leads to aromatization of the A-ring accompanied by cleavage of the B-ring, and aromatized A-ring is hydroxylated at C-4 position, cleaved at Δ4 by meta-cleavage, and divided into 2-hydroxyhexa-2,4-dienoic acid (A-ring) and 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid (B,C,D-ring) by hydrolysis. Reactions and the genes involved in the cleavage and the following degradation of the A-ring are similar to those for bacterial biphenyl degradation, and 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid degradation is suggested to be mainly β-oxidation. Genes involved in A-ring aromatization and degradation form a gene cluster, and the genes involved in β-oxidation of 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid also comprise a large cluster of more than 10 genes. The DNA region between these two main steroid degradation gene clusters contain 3α-hydroxysteroid dehydrogenase gene, Δ5,3-ketosteroid isomerase gene, genes for inversion of an α-oriented-hydroxyl group to a β-oriented-hydroxyl group at C-12 position of cholic acid, and genes possibly involved in the degradation of a side chain at C-17 position of cholic acid, indicating this DNA region of more than 100kb to be a steroid degradation gene hot spot of C. testosteroni. Article from a special issue on steroids and microorganisms.     

Synthesis and molecular structure of 14,15-pyrrolidino-and 14,16-ethano derivatives of estrone

Hydrolysis of 3-methoxy-16alpha-nitro-14,17-ethenoestra-1,3,5(10)-trien-17beta-yl acetate under weakly basic conditions leads to formation of 3-methoxy-2'-oxopyrrolidino-[4',5':14beta,15beta]-estra-1,3,5 (10)-trien-17-one, the structure of which has been confirmed by X-ray analysis and some chemical transformations. The reactivity of 3-methoxy-16alpha-nitro-14,17-ethanoestra-1,3,5(10)-trien-17beta-yl acetate under various conditions of basic hydrolysis has been investigated. The derived compounds have been identified by means of NMR spectroscopy and X-ray analysis.     

Omega-pyridiniumalkylethers of steroidal phenols: new compounds with potent antibacterial and antiproliferative activities

Novel omega-pyridiniumalkylethers of two steroidal phenols were synthesized as compounds with potential antimicrobial activity. 3-Hydroxy-estra-1,3,5(10)-triene-17-one and 1-hydroxy-4-methyl-estra-1,3,5(10)-triene-17-one were reacted with omega,omega'-dibromoalkanes to omega-bromoalkoxy-estra-1,3,5(10)-trienes followed by reaction with pyridine to obtain the desired steroidal omega-pyridiniumalkoxy compounds as bromides. Their antimicrobial activity against strains of multiresistant Staphylococcus aureus (MRSA), a vancomycin resistant Enterococcus faecalis and fast growing mycobacteria depends clearly on the length of the alkyl chain. A strong broadband activity has been found for the compounds with eight or 10 C-atoms; in some cases better than ciprofloxacin or cetylpyridinium salts. In addition, the antiproliferative and cytotoxic activity depends on the chain length, too. The differentiation between antibacterial and cytotoxic activity is better for the steroid hybrid molecules than the cetylpyridinium salts. These new compounds can serve as lead compounds for further optimization.     

The aromatization of (7 -3H) androstenedione by human placental mitochondria

A metabolite of 2,3-dihydroxyestra-1,3,5(10)-trien-17-one-6, 7-³H isolated from rat bile, was partially characterized by mass spectrometry as a methyl ether of 2,3,16-trihydroxyestra-1,3,5(10)-trien-17-one. The α configuration of the 16-hydroxy function was established by chromatographic comparison of the sodium borohydride reduced metabolite with synthetic 2-methoxy-estra-1, 3,5(10)-triene-3,16α,17β-triol and 2-methoxy-estra-1, 3,5(10)-triene-3,16β,17β-triol. The methyl group was located on the C-2 position by comparison with authentic 2- and 3- monomethyl ethers of 2,3-dihydroxy-estra-1, 3,5(10)-trien-17-one following pyrolytic removal of the 16α-hydroxyl group.3,16α-dihydroxy-2-methoxyestra-1,3,5(10)-trien-17-one was found to constitute 2% and 15% of the biliary radioactivity following administration of estrone-6,7-³H and 2,3-dihydroxyestra-1,3,5(10)-trien-17-one-6,7-³H respectively.     

Etienic etienate as synthon for the synthesis of steroid oligoester gelators

Linear oligoesters based on etienic acid (3beta-hydroxyandrost-5-ene-17beta-carboxylic acid) containing four steroid units were prepared using a 2+2 synthetic strategy in a successful synthesis of 3beta-{[3beta-({3beta-[(3beta-hydroxyandrost-5-ene-17beta-carbonyl)oxy]androst-5-ene-17beta-carbonyl}oxy)androst-5-ene-17beta-carbonyl]oxy}androst-5-ene-17beta-carboxylic acid. The main problems with deprotection were overcome by using orthogonal groups as O-nitrates and 2-(trimethylsilyl)ethyl ethers.     

Detection of a transient enzyme-steroid complex during active-site-directed irreversible inhibition of 3-oxo-delta 5-steroid isomerase

The reaction of the active-site-directed irreversible inhibitor (17S)-spiro[estra-1,3,5(10),6,8-pentaene-17,2'-oxiran]-3-ol (5 beta) with 3-oxo-delta 5-steroid isomerase has been monitored by repetitive scanning ultraviolet spectroscopy of a solution of 5 beta plus isomerase against a blank containing only 5 beta. Upon initial mixing of 5 beta with the isomerase an absorbance maximum at ca. 250 nm appears. With time, this peak decreases and is replaced with a new peak near 280 nm. These results directly demonstrate the existence of a transient enzyme-steroid intermediate in the inactivation reaction. The ultraviolet spectrum suggests that the steroid in the transient complex resembles the ionized phenol, while the phenolic group in the irreversibly bound complex is un-ionized. These spectral studies support our previous proposal that there are two enzyme-steroid complexes that are related by a 180 degree rotation about an axis perpendicular to the plane of the steroid nucleus. This hypothesis offers an explanation for the reaction of 17 beta-oxiranes with the same residue (Asp-38) that is thought to be involved in the catalytic mechanism. Two new oxiranes, (17S)-spiro[estra-1,3,5(10)-triene-17,2'-oxiran]-3 beta-ol (6 beta) and (17S)-spiro[5 alpha-androstane-17,2'-oxiran]-3-one (8 beta), were also found to be potent active-site-directed irreversible inhibitors of the isomerase (k3/KI = 31 M-1 s-1 and 340 M-1 s-1, respectively). The relationship of these results to the nature of the active site of the isomerase is discussed.     

Efficient syntheses of 17-β-amino steroids

17β-Amino steroids such as 17β-amino-1,3,5(10)-estratrien-3-ol (1), 17β-amino-5α-androstan-3β-ol (2) and, 17β-amino-3β-hydroxyandrost-5-ene (3) have been widely used as a key intermediates in the synthesis of a variety of biologically active steroid derivatives though concise, high yielding syntheses of these compounds has yet to be reported. 17β-Amino-1,3,5(10)-estratrien-3-ol (1) and 17β-amino-5α-androstan-3β-ol (2) were prepared in high yield by reductive amination of estrone and epiandrosterone using benzylamine and sodium triacetoxyborohydride followed by catalytic hydrogenolysis of the resulting 17β-benzylamino derivatives. Attempts to prepare 17β-amino-3β-hydroxyandrost-5-ene (3) from dehydroepiandosterone using a similar approach resulted in partial reduction of the double bond. 17β-Amino-3β-hydroxyandrost-5-ene (3) was ultimately obtained in high yield by reductive amination of dehydroepiandosterone using allylamine and sodium triacetoxyborohydride followed by removal of the allyl group from the resulting 17β-allylamino derivative with dimethylbarbituric acid and Pd(PPh(3))(4) as catalyst.     

Isolation of histidyl peptides of the steroid-binding site of human placental estradiol 17β-dehydrogenase

Human placental estradiol 17β-dehydrogenase (E.C. 1.1.1.62) was inactivated at pH 6.3 by 3-bromo[2′-¹⁴C]acetoxy-1,3,5(10)estratrien-17-one, a known substrate. The affinity-alkylated enzyme was then hydrolyzed by trypsin. Radioactive peptides were initially isolated by gel filtration and identified according to which residue was alkylated. Tryptic peptides containing radioactive 3-carboxymethylhistidyl residues were further purified by cation-exchange chromatography. The population of these peptides varied, depending upon the conditions of enzyme inactivation. With 60 μM 3-bromo[2′-¹⁴C]acetoxy-1,3,5(10)estratrien-17-one four major peptides (a,b,c,d) each containing radioactive 3-carboxymethylhistidine, were eluted from the cation-exchange column. The alkylation of all of these peptides was completely suppressed when the enzyme was inactivated in the presence of excess estradiol-17β. The presence of equimolar NADPH during incubation greatly enhanced the alkylation of all four peptides. In the presence of NADPH, estradiol-17β most significantly decreased the formation of peptide d. Peptide d was the only peptide identified when the concentration of the alkylating steroid was lowered to 6 βM, a value approaching the Km. These observations indicate that peptide d is a histidyl-bearing peptide from the steroid-binding site which proximates the steroid A-ring. They further suggest that with the affinity labeling steroid at higher concentrations other nonspecific, hydrophobic sites on the enzyme are occupied and labeled.