Mechanism of Androstenedione Formation from Testosterone and. Epitestosterone Catalyzed by Purified Cytochrome P-450b*. (Received for publication, March ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 263, No. 33, Issue o f November 25, pp. 17322-17332,1988 Printed in U.S.A.
Mechanism of Androstenedione Formation from Testosteroneand Epitestosterone Catalyzed by Purified Cytochrome P-450b* (Received for publication, March 28, 1988)
Alexander W. Wood$§, David C. SwinneyllII,Paul E. Thomasll, Dene E. Ryanll, Peter F. Hall**$$, Wayne Levinll, and William A. Garland§§ From the Departments of $Oncology and Virology, (Protein Biochemistry, and @Drug Metabolism, Hoffmann-La Roche Inc., Nutley, New Jersey 07110 and **The Worcester Foundationfor Experimental Biology, Shrewsbury, Massachusetts 01545
A purified rat hepatic monooxygenase system conto a number of hydroxylated products of unknown biological tainingcytochrome P-450b oxidizestestosteroneto activity (2,3). These oxidations, when catalyzed by individual androstenedione and 16a- and 168-hydroxytestoster- highly purified hepatic cytochrome P-450 isozymes, are markone at approximately equal rates. The metabolism of edly regioselective (4-6). epitestosterone bythe same system is characterized by This paper reports results of experiments using stable isoa marked stereoselectivity in favor of 168-hydroxyl- tope methodology to characterize the metabolism of testosation (4- to&fold relative to 16a-hydroxylation), for- terone’ and its 17-hydroxy epimer, epitestosterone, by cytomation of 15a-hydroxyepitestosterone,and a rate of chrome P-450b,’ the major hepatic microsomal protein inandrostenedione formation whichis three to five times duced by phenobarbital (8).The study was undertaken with higher than that observed with testosterone. Apparent the goals of (i) further defining the effect of steroid structure K,,, values for 16a- and 168-hydroxylation and androstenedione formation are 20-30 p~ with either sub- on stereoselective product formation catalyzed by cytochrome strate. Mass spectral analysis of the androstenedione P-450b, and (ii) elucidating the mechanism by which cytoformed from [16,16-2H2Jtestosterone and [16,16-2H2J chrome P-450b catalyzes the c-17 oxidation of testosterone and epitestosterone to form androstenedione. This reaction is epitestosterone indicates essentially complete retention of deuterium, thereby ruling outa mechanism of unique in that the monooxygenase system catalyzes an oxiandrostenedioneformationvia C- 16 hydroxylation dative reaction without the net incorporation of oxygen into followed by loss of water and rearrangement. Mass the product. Of the 11 cytochrome P-450 isozymes studied to date, Pspectral analysis of the C-16 hydroxylation products from incubations of testosterone or epitestosterone in 450b has the highest capacity (V,,,) to catalyze androstene“ 0 2 shows essentially complete incorporation of ‘*O dione formation from testosterone (cf. Ref. 6). Our previous (>%YO). Androstenedione formed from testosterone is studies indicated that androstenedione formation from tesenriched in “0 only 2-fold (5-8%)over background, tosterone was totally dependent on NADPH, NADPH-cytowhile the androstenedione formed from epitestosterchrome P-450 reductase, and the cytochrome and could not one shows 84% enrichment. Kinetic experiments utibe attributable to a NADP-dependent dehydrogenation (4). lizing [ 17-2H]testosterone and [ 17-2H]epitestosterone In addition, monospecific polyclonal and monoclonal antibodas substrates indicate that cleavage of the C- 17carbon- ies directed against cytochrome P-450b totally inhibited the hydrogen bond is involved in a rate-limiting step in cytochrome P-450b-catalyzed formation of androstenedione the formation of androstenedione from both substrates.from testosterone in the reconstituted system as well as Taken together, our results indicate that androstene- hepatic microsomes from phenobarbital-treated rats (9).3 dione formation from epitestosteroneproceeds exclusively through the gem-diol pathway, while androEXPERIMENTALPROCEDURES stenedione formation from testosterone may proceed through a combination of gem-diol and dual hydrogen Instrumentation abstraction pathways.
Cytochrome P-450-dependent reactions in endocrine tissues catalyze the synthesis of steroid hormones from cholesterol (l),while the cytochrome P-450-dependent monooxygenases in the endoplasmic reticulum of liver oxidize steroids
Chemical ionization mass spectra were obtained using a Finnigan 1015 GC/MS equipped with a Finnigan 9500 GC and a Finnigan 6000 data system with revision I software (Finnigan Instrument Corp.). The glass GC column, 1.5 m long with a 2-mm inner diameter, was packed with 3%SE-30 on 100-120 mesh GCQ (Applied Science Laboratories) and was operated at 250 “C. The methane carrier gas pressure was 1 kg m-*, and the interface oven and transfer line were operated at 200 “C. Under these conditions, the retention time of
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § To whom all correspondence should be addressed Dept. of Oncology and Virology, Hoffmann-La Roche Inc., Bldg. 86, Nutley, NJ 07110. 11 Present address: Dept. of Drug Metabolism, Syntex Corp., Palo Alto, CA 94303. $$ Present address: Dept. of Endocrinology, Prince of Wales Hospital, University ofNew South Wales, Randwick, 2031 NSW, Australia.
’The trivial names and abbreviations used are: testosterone, 17P-hydroxy-4-androsten-3-one; epitestosterone, 17a-hydroxy-4-androsten-3-one; androstenedione, 4-androstene-3,17-dione; 17-desoxytestosterone, 4-androsten-3-one; HPLC, high pressure liquid chromatography, OHT, hydroxytestosterone; GC/MS, gas chromatography-mass spectrometry; EIMS, electron ionization mass spectrometry; CIMS, chemical ionization mass spectrometry. * According to a recently recommended nomenclature system for cytochrome P-450s (7), ratcytochrome P-450b is encoded by rat gene IIB1. P. E. Thomas, D. E. Ryan, L. M. Reik, and W. Levin, unpublished observations.
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Cytochrome P-450b-dependent Oxidation Androgen
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with 3.4mgof NaBH4. Water (0.1 ml) was added, and the stirring was continued for another 30 min. Sufficient 10 N HC1 was added to make the solution pH 1, and stirring was continued for 3 h, at which time the solution was extracted with ethyl ether (3 X 15 ml). The ethyl ether was dried over anhydrous MgS04 and filtered, and the ether was removedunder a streamof dry Nz. The resulting oil droplets (2.1 mg, 7%) were dissolved in methanol, and aliquots were chromatographed using the HPLC system described below for the synthesis of [16,16-2H]testosterone. The effluents atthe retention volume corresponding to authentic testosterone were collected and, aided by successive additions of ethanol, were evaporated to dryness under a stream of dry Nz. The residue had suitable chromatographic and spectroscopic properties, and the label was exclusively located at C17 as determined by EIMS and NMR. GC/CIMS analysis indicated Chemicals the compound was 55% l80and 45% unlabeled. Testosterone, deuterium oxide(99.8 atom %), tetradeuterated [16,16-2H]Testosterone and [16,16-2HJEpitestosterone-A sliver methanol (99.5 atom %), CH30'H (99.5 atom %), and 2,2-dimethox- of sodium was added to 5 ml of tetradeuterated methanol. Following ypropane were purchased from Aldrich; epitestosterone, androstene- evolution of hydrogen gas, 3-methoxy-3,5-androstadiene-l7-one (600 dione, and 1601-hydroxytestosteronewere purchased from Steraloids, mg, 2 mmol) was added, and the solution was transferred to a 25-ml Inc.; 16-keto-17-desoxytestosteronewas purchased from Research glass ampule which was sealed and was placed in an oven set to Plus, Inc. 17-Desoxytestosterone and 1601-hydroxy-17-desoxytestos- 100 "C. After 1 day, the ampule was unsealed, and the solvent was terone were gifts of the Steroid Reference Collection, Medical Re- removed under a stream of dry Nz. The residue was reconstituted in search Council, London. tetradeuterated methanol (5 ml) and the treatmentdescribed above, Catalase, superoxide dismutase, D-mannitol, NAD, NADP, and except for the addition of sodium, was repeated. The residue from the NADPH were obtained from Sigma. Cumene hydroperoxide was second treatment was extracted with ethyl ether (3 X 10 ml) and purchased from ICN Laboratories; hydrogen peroxide (30%),D-glu- filtered, and thecombined extracts were evaporated to dryness under cose, and all inorganic chemicals were purchased from Fisher; glucose a stream of dry NZ.The residue, [16,16-2Hz]3-methoxy-3,5-androstaoxidase was purchased from Boehringer Mannheim; and dilauroyldiene-17-one, was dissolved in dry methanol (5 ml) and treated with phosphatidylcholine was obtained from Behring Diagnostics. Oxygen NaBH4 in a manner identical to that described for the synthesis of gas (98 atom % was obtained from Cambridge Isotope Labora[17-zH]testosteroneand [17-2H]epitestosterone. The resulting white tories, Inc. Deuterochloroform (99.996 atom %) and HZl80 (97.4 atom powder was dissolved in methanol (2 ml), and 50-pl aliquots were %) were obtained from MSD Isotopes. Sodium borotetradeuteride (99 chromatographed on a Magnum-10 HPLC column (Whatman) with atom %), LiA1HZ4(99 atom %), and ['Hslhydrazine hydrate (98 atom methanol/water/acetonitrile (90:5:5). The peaks eluting at 19 min %) were purchased from Stohler Isotopes. Regisil@was purchased (testosterone) and 24 min (epitestosterone) were collected, and the from Regis. Sodium borotetrahydride was purchased from Ventron. solvents were removed under vacuum. The resulting white powders 3-Methoxy-3,5-androstadiene-l7-one was synthesized using the (160 mg of testosterone, 27% yield,and 4 mg of epitestosterone, 0.7% method of Nussbaum et al. (10).All gases used in the GC/MS analyses yield) had suitable spectroscopic properties. By GC/CIMS (silylated wereof the highest quality available from Liquid Carbonic. All derivative), the testosterone was 95% 'Hz, 3% 'H, and 2% unlabeled. solvents, except ethyl ether,were of the highest quality available from The epitestosterone was 91% 'Hz, 7% 'H, and 2% unlabeled. Burdick and Jackson. Ethyl ether was from Mallinckrodt. All pre[16,16,17,1 7-2HJDesoxytestostero~-The title compound was parative TLC was carried out using 2-mm silica plates from L. Merck. synthesized using a modification of the Wolf-Kishner procedure described by Patterson et al. (11).Diethylene glycol (20 ml) was Synthesis refluxed with 'Hz0 (20 ml) for 1 h. The temperature was increased [ l 7-2H]Testosterone and [l 7-ZH]Epitestosterone-A solution of 3- to 180 "C and the 'Hz0 was removed using a Dean-Stark trap. The methoxy-3,5-androstadiene-17-one (300 mg, 1 mmol) in 5 ml of dry remaining solution was cooled, another 20 ml of Hz0 was added, and methanol was cooled to 2 "C and was subjected, with stirring, to two the reflux and 'Hz0 removal procedures were repeated. After cooling successive treatments with NaB2H4(38 mg, 1 mmol), 30 min apart. the solution to room temperature, sodium (200 mg) and [16,16-2H~] Water (0.5 ml) was added, and thestirring was continued for another 3-methoxy-3,5-androstadiene-17-one (60 mg, 0.2 mmol) were added. 30 min. Sufficient 10 N HCl was added to make the solution pH 1, The solution was heated to 180 "C, and ['Hslhydrazine (0.5 ml, 15.8 and after stirring for three hours, the solution was extracted with mmol) was added. The reflux condenser was removed, and the temethyl ether (3 X 15 ml). The ethyl ether was dried over anhydrous perature was allowed to rise to 210 "C. Heating at reflux was continMgS04, filtered, and was then evaporated at room temperature under ued at this temperature for 6 h. The mixture was then poured into a stream of dry Nz. The resulting powder was dissolved in 1 ml of water and was extracted with ethyl ester (3 X 50 ml) followed by methanol, and aliquots were chromatographed using the HPLC sys- washing (3 X 10 ml of HZO), drying (MgSOI), and evaporation to give tem described below for the synthesis of [16,16-2H]testosterone and a yellow oil. The oil was dissolved in methanol (10 ml) and 10 N HCl [16,16-2H]epitestosterone.The effluents at the retention volumes of (1 ml). After 3 h, the solvents were removed under a stream of Nz, authentic testosterone and authentic epitestosterone were collected and the residue was chromatographed by preparative TLC (hexane/ and, aided by successive additions of ethanol, were evaporated to ethyl acetate, 8 2 ) . The band at RF = 0.6 was scraped and was eluted dryness under a stream of dry N,. The residues had suitable chro- with ethyl acetate. The powder remaining after removal of the ethyl matographic and spectroscopic properties, and the labels were exclu- acetate had suitable chromatographic and spectral properties. By sively located at C-17 as determined by EIMS and NMR. GC/CIMS GC/CIMS, the compound was 12% 'Ha, 75% 'H4, and 13% 'H5. analysis of the silylated derivative indicated that thetestosterone was 160-0H Desorytestosterone-16-Ketodesoxytestosterone (143 mg, 89% monodeuterated and 11%unlabeled, and that the epitestosterone 0.5 mmol) was dissolved in 2,2-dimethoxypropane (5 ml) and dimethwas 90% monodeuterated and 10% unlabeled. ylformamide (5 ml). Methanol (10 pl) andp-toluene sulfonic acid (10 I1 7-'801Testosterone-A sliver of sodium was added to a solution mg) were added, and the solution was refluxed for 8 h. After cooling, of 1 mlofH2180 and 10 mlof methanol. Following evolution of the solution was neutralized with 100 mg of NaHC03, water (15 ml) hydrogen gas, 3-methoxy-3,5-androstadiene-l7-one (30 mg, 0.1 mmol) was added, and the solution was extracted with ethyl ether ( 2 X 50 was added, and the solution was transferred to a 25-ml glass ampule ml). The ether was removed under a stream of Nz, the oily residue which was sealed and placed in an oven set to 110 "C. After 1 week, was reconstituted in methanol (1 ml) and chromatographed by TLC the ampule was unsealed, and the solvents were removed at 0.1 torr (hexane/ethyl acetate, 8:2). The band at RF = 0.7 was scraped and in a rotary evaporator equipped with a dry ice trap. The residue was eluted with ethyl acetate. The oil remaining after removal of the ethyl reconstituted in 10 ml of fresh methanol and 1 ml of HZ"0, and the acetate was reduced with NaBH4, and the enol ether was hydrolyzed treatment described above, without the addition of sodium, was with 10 N HCl as described previously for the preparation of [ 17-'H] repeated. The residue from the second weekof treatment was ex- testosterone and [17-2H]epitestosterone. The residue from the final tracted with ethyl ether (3 X 10 ml) and filtered, and the combined ethyl ether extraction was dissolved in ethyl acetate and chromatoextracts were evaporated to dryness under a stream of dry Nz. The graphed by TLC (hexane/ethyl acetate, 7:3). The band at RF = 0.2 residue was dissolved in dry methanol (5 ml), andthe stirred solution was scraped and was eluted with ethyl acetate. The white powder (4 was cooled to 2 "C before two successive treatments, 30 min apart, mg), remaining after removal of the ethyl acetate, had suitable mass
silylated testosterone was approximately 3 min. Isobutane (0.2 torr) was the chemical ionization reagent gas. Electron ionization mass spectra (70 eV) wereobtained using a Vg ZAB-1F instrument with a Vg2025 data system. Samples were introduced using the direct insertion probe inlet. Proton NMR spectra were obtained with a Varian XL-400 spectrometer operated in the Fourier transform mode. Tetramethylsilane was the internal reference, and the solvent was CDCl,. The pulse width was equivalent to a 30" flip angle, and thepulse repetition rate was 2 s. The probe temperature was 21 "C. The HPLC instrumentation and the analytical conditions have been described (4).
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Cytochrome P-450b-dependent Androgen Oxidation
spectral and NMR properties (EIMS (m/z > 120 and 20% relative abundance): m/z 288 (M?, 30%), m/z 270 (M? - HzO,80%), m/z 255 ( m / z 270-CH3,40%),m/z 147 (CJIIEO, 100%) and m/z 124 (C8Hlz0, 75%); NMR shows signals a t 64.41 (M, lH, HO-C-E), 1.19 (S, 3H, C-18 methyl), and 61.00 (S, 3H, C-19 methyl)).
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Enzyme Preparations Cytochrome P-45Obwas purified to homogeneity from hepatic microsomes of Aroclor 1254-treated immature male Long Evans rats as previously described (12). NADPH-cytochrome P-450 reductase was purified from hepatic microsomes of phenobarbital-treated immature male Long Evans rats to a specific activity of 35,000-40,000 units/mg protein by a modification (13) of the methods of Yasukochi and Masters (14) and Dignam and Strobe1 (15). One unit of reductase catalyzes the reduction of 1 nmol of cytochrome c/min at 22 “C in 0.3 M potassium phosphate buffer (pH 7.7) containing 0.1 mM EDTA and 0.1 mM NADPH. Incubation, Extraction, and Analytical Conditions The standard incubation conditions for the metabolism of the steroids were as previously described in detail (4),and the extent of metabolism was always proportional to the concentration of cytochrome P-450b and time of incubation. Briefly, incubations generally contained from 0.025 to 0.10 nmol of cytochrome P-450b, 1200 units of NADPH-cytochrome e reductase, 10 pg of dilauroylphosphatidylcholine, 1 kmol of NADPH, 3 pmol of magnesium chloride, 50 pmol of potassium phosphate buffer (pH 7.4), and 125 nmol of steroid (added in 20 p1 of methanol) in a final volume of 1.0 ml. Incubations were performed by preincubating the samples for 2 min atthe appropriate temperatures (15-47 “C) before starting the reactions with the addition of NADPH. Incubations with “OZwere performed in screw-capped, Teflon@” lined, 15 X 125-mm tubes equipped with a glass side arm. The main chamber of the tube contained allthe incubation components except the solution of NADPH (0.05 ml), which was placed in the side arm. Buffers were boiled and were stored under an atmosphere of argon prior to use. Reaction mixtures were made anaerobic by alternatively evacuating (80 torr) and flushing the tubes with argon a total of seven times. During the final three cycles, the incubation mixtures were shell-frozen and were thawed under vacuum. After flushing the system twice with 0 2 or “ 0 2 and equilibrating the mixtures for 2 min a t the desired temperature, the reaction was started by introduction of NADPH from the side arm. The incubation was stopped by rapid freezing in an acetone/dry ice bath. Substrate and metabolites were extracted quantitatively in 6 volumes of methylene chloride. Following evaporation of solvent under dry Nz, the sample residues were dissolved in methanol and were analyzed by HPLC as previously described (4) using a 5-pm octyldecylsilane reverse phase column (Supelco, Inc.) and a linear gradient from 43% methanol, 55.9% water, and 1.1%acetonitrile to 75% methanol, 23.1% water, and 1.9% acetonitrile at a flow rate of 1.5 ml/min. For NMR and MS analyses, the eluate fractions corresponding to each metabolite were combined and were evaporated to dryness (with the aid of successive additions of ethanol) under a stream of dry Nz. For NMR analyses, the residues were rechromatographed using a C18 pBondapak HPLC column (Waters) and methanol/water (80:20). For electron ionization mass spectral analyses, the residues were dissolved in 100 pl of methanol, and 20-30 pl were applied to thetip of the direct insertion probe by successive evaporations of 5-p1 aliquots. For chemical ionization GC/MS analyses, the residue was solution was allowedto stand dissolved in 100 p1 of Regid@, and the overnight at 70 “C in a capped tube. Five p1of this solution was injected into the GC/MS.
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FIG. 1. HPLC profiles of the metabolites formed from incubating the purified monooxygenase system, reconstituted with 0.10 nmol of cytochrome P-460b,with 126 M M testostertrace A ) , 126 p~ epitestosterone (Epi T,trace B ) , or one (T, 36 p~ desoxytestosterone (Desoxy T,trace C)for 6,3,and 1 min, respectively. Extraction residues were prepared and dissolved in methanol as described under “Experimental Procedures,” and the volume of methanol solution was injected into the HPLC, adjusted so that the most prominent metabolite of each substrate gave approximately 95% full-scale absorbance. Androstenedione is abbreviated A , and with the exception of 16p-OHA (16p-hydroxyandrostenedione) and 16 = 0 (16-ketodesoxytestosterone)the abbreviations represent monohydroxy derivatives of the parentsteroid.
structure of 16/3-hydroxyandrostenedione (4). Confirmation of the identity of this metabolite was provided by incubation of deuterated analogs of testosterone. The GC/MS analysis of the peak following incubation with testosterone showed a silylated metabolite having a molecular weight of 374 (silylated testosterone plus one ketone function). The silylated metabolite following incubation of [17-2H]testosterone also had a molecular weight of 374, suggesting the keto function RESULTS was at C-17. The silylated metabolite following incubation of Identification of Metabolites [16,16-2H2]testosteronehad a molecular weight of 375, suggesting the hydroxy function was at C-16. Testosterone-A typical chromatogram of testosterone meEpitestosterone-A typical HPLC chromatogram of epitestabolism catalyzed by cytochrome P-45Ob in a reconstituted system is shown in Fig. l.4. As previously reported (4), cyto- tosterone metabolism catalyzed by cytochrome P-450b is chrome P-450b catalyzed the oxidation of testosterone a t the shown in Fig. 1B. Metabolite formation was completely deC-16 and (2-17 positions to yield 16a-hydroxytestosterone, pendent on cytochrome P-450b, NADPH-cytochrome P-450 16/3-hydroxytestosterone, and androstenedione in approxi- reductase, and NADPH. Neither NAD or NADP could supmately equal molar ratios. The peak in the chromatogram port the reactions, Anaerobic incubations in an argon atmoswith a retention time of 7 min was previously assigned the phere inhibited metabolism by over 90%, and monoclonal
Cytochrome P-450b-dependent Androgen Oxidation antibodies directed against two distinct epitopes on cytochrome P-450b (9) completely inhibited the formation of all metabolites (data not shown). The two major metabolites eluting at 8.1 and 13.4 min were identified as 16P-hydroxyepitestosterone and androstenedione, respectively. Identification of the latter metabolite as androstenedione was based on the fact that its chromatographic and spectral properties were identical to authentic standard. Following silylation, the other major metabolite (168-hydroxyepitestosterone) had amolecular weight of 448 (silylated epitestosterone plus one silylatable hydroxy function) as determined by the GC/MS analysis. Consistent with these data, the M+ ion in the electron impact mass spectrum of the metabolite was at m/z 304. The key m/z 147 fragment ion (16) was shifted to m/z 163, suggesting that hydroxylation had occurred on the C- or D-rings of the steroid. The analogous silylated metabolite following incubation of [16,16-'H~] epitestosterone had a molecular weight of 449 suggesting that the hydroxy was at C-16. Compared to the NMR spectrum of epitestosterone, the signal at 63.79 (d, lH, J = 5 Hz, H-17) in the spectrum of the metabolite collapsed to a singlet at 3.65 (S, lH, H-17), and a new signal appeared at 64.11 (t, lH, J = 7 Hz). In this regard, the signal from H-17 was a doublet in the spectrum of epitestosterone, and not a triplet as in the spectrum of testosterone, because the dihedral angle in epitestosterone between H-17 and one of the c-16 hydrogens is 90". Since this signal was not a singlet, it suggested by the same reasoning that the hydrogen remaining after hydroxylation was perpendicular to H-17, i.e. the metabolite was 16Phydroxyepitestosterone. In addition, the triplet at 64.11 was expected due to coupling of H-16 with the two hydrogens at C-15. A P-configuration was also suggested by the chemical shift of the C-18 methyl resonance from 60.72 in the spectrum of epitestosterone to 60.95 in the spectrum of the metabolite (17). The threeminor metabolites that eluted at 7,9, and13 min were identified as 16P-hydroxyandrostenedione,15a-hydroxyepitestosterone, and 16a-hydroxyepitestosterone, respectively. Following silylation, the first peak had a molecular weight of 374 (silylated testosterone plus one ketone function), as determined by the GC/MS analysis. The silylated metabolite following incubation of [ 17-'H]epitestosterone also had molecular weight of 374, suggesting that the keto is at C-17. The silylated metabolite following incubation of [16,l6-'Hz]epitestosterone had a molecular weight of 375, suggesting that the hydroxy was at C-16. The NMR showed a singlet at 63.77((2-16 proton) and no other signal in the range expected for E-C-OH. Finally, the metabolite had the same retention time as 16P-hydroxyandrostenedionegenerated from testosterone. The peaks eluting at 9 and 13min both had EIMS andGC/ MS (silylated derivative) properties consistent with monohydroxylated metabolites. With incubation of [ 16,16-2Hz]epitestosterone, the first eluting metabolite retained both deuteriums, while the second eluting metabolite lost one, i.e. hydroxylation at C-16. The NMR of this latter compound showed a doublet at 3.67 (H-17) and a multiplet at 4.50 (EC-OH at C-16). Consistent with the proposed configuration was the fact that themethyl resonances at C-18 were relatively unaffected, i.e.60.76,by the hydroxylation. Since the H-17 doublet was undisturbed, the hydroxylation must be at the hydrogen 90" to the hydrogen at C-17, i.e. a-hydroxylation and themetabolite eluting at 13 min is 16a-hydroxyepitestosterone. Consistent with the proposed structure for the metabolite
17325
eluting at 9 min (15a-hydroxyepitestosterone),the E1 mass spectrum showed an ion at m/z 163 (no m/z 147)which located the hydroxylation on the C- or D-ring. By elimination, the hydroxylation could not be at C-16. A small ion at m/z 215 in the spectrum was best explained by loss of the D-ring along with the hydroxy substituent at C-15 as shown in Scheme 1. Supporting this mechanism are the facts that Hz0 (and not 'H20 or 'HHO) was lost in the E1 mass spectra of [17-'H]and [ 16,16-*H2]testosteroneand epitestosterone, andthat an ion in the spectrum was also observed at m/z 229. However, this latter ion may also have been generated from the loss of methyl radical ion from the ion at m/z 244 (M?-HzC=C=OH20).The NMR of the metabolite showed signals at 60.77 ( S , C-18 methyl), 63.50 (d, C-17 hydrogen), and 64.03 (M, E-COH). Based on the relatively small methyl shift compared to epitestosterone, the configuration of the hydroxy group is a. Based on studies with testosteroneand progesterone, the chemical shift of the hydrogen at C-15 would be expected to be at approximately 64.1 (18).The upfield shift of the H-17 proton from 63.77 in epitestosterone to 63.50 in the metabolite continues a trend also observed for D-ring hydroxylated metabolites of testosterone. 17-Desoxytestosterone-The metabolism of 17-desoxytestosterone was primarily investigated to determine whether cytochrome P-450b could catalyze carbon-oxygen bond formation at (2-17. The chromatographic profile in Fig. 1C clearly indicates that neither androstenedione nor epitestosterone were formed.The principal metabolite peak had chemical and electron ionization mass spectra and retention times by GC and HPLC virtually identical to those of testosterone. However, the NMR spectrum was quite different from that of testosterone, i.e. the C-18 methyl signal was shifted from 60.79 (S, 3H, CH3-18) to 1.00 and, instead of the signal at 63.56 (t, lH,J = 9 Hz, H-17), there was a complex multiplet at 64.41. The silylated metabolite following incubation of [ 17,17,16,16-2H4]17-desoxytestosteronehad a molecular weight of 364, suggesting that thehydroxylation was at C-16. Based on this observation, 16P-hydroxy-17-desoxytestosterone was synthesized from authentic 16-keto-17-desoxytestosterone. The synthesized material had a NMR spectrum and a HPLC retention time identical to those of the metabolite. The minor peak eluting at 16.4 min in Fig. 1C was identified as 16a-hydroxy-17-desoxytestosteroneby mass spectral analysis and cochromatography with authenticstandard, while the minor peak eluting at 14.1 min was assigned the structure of 16-keto-17-desoxytestosteronebased on mass spectral analysis and cochromatography with authentic standard.Both metabolites showed deuterium losses consistent with the above structure assignments when [17,17,16,16-2H4] 17-desoxytestosterone was used as substrate. Time course experiments indicated that formation of the 16-keto metabolite lagged behind the formation of the other two metabolites. It was also observed that thecytochrome P450b-dependent monooxygenase system oxidized both 16ato the16-keto metaband 16~-hydroxy-17-desoxytestosterone olite (data not shown), analogous to the conversion of 16phydrotestosterone to 16-ketotestosterone (4). Kinetics of Metabolite Formation The kinetic constants associated with the reactions just described are summarized in TableI. The apparentK,,, values for the formation of the three primary oxidative metabolites of testosterone catalyzed by cytochrome P-450b were essentially identical (25-28 FM), and the VmaXvalues were comparable to turnover numbers observed with a saturating (125
17326
Cytochrome Pd5Ob-dependent Oxidation Androgen
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H2C"H H2C"H
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on m h 228
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SCHEME 1 TABLE I Kinetic constants for the metabolism of testosterone, epitestosterone, and 17-desoxytestosteroneby a purified monooxygenme system reconstituted with cytochromeP-450b Kinetic constants were determined from slope and intercept values obtained by unweighted least squares curve fits of Lineweaver-Burk double-reciprocal plots of initial velocities versus substrate concentration. At least eight concentrations of each substrate, covering an 8- to 16-fold concentration range, were utilized, and P values for the linear curve fits ranged from 0.93 to 0.96. Amounts of cytochrome P450b in the incubation mixture and times of incubation were adjusted such that no more than 17% of the substrates were metabolized. Any further metabolism of androstenedione to 160-hydroxyandrostenedione has been accounted for in the determinations of the rate of androstenedione formation. Metabolite Km,.pp) VIM, Substrate
could never be definitively demonstrated when testosterone was the substrate, although formation of the metabolite at 5% of the rate observed for the 15a-hydroxylation of epitestosterone would have been readily detected. 17-Desoxytestosterone was an exceptionally good substrate for cytochrome P-450b as illustrated by the apparent K, of 0.3 PM (Table I), which was almost 2 orders of magnitude lower than thevalues observed with testosterone or epitestosterone. The Vmaxvalue for the 16P-hydroxylation of 17-desoxytestosterone was five and 12 times higher than the rates observed for the lG@-hydroxylationof epitestosterone and testosterone, respectively (Table I).
Effect of Active Oxygen Scavengers on Metabolite Formation Neither catalase (144 units/ml), superoxide dismutase (280 nmolfminf units/ml), mannitol (50 mM), nor dimethyl sulfoxide (30 mM) pM nmol P-450 inhibited the metabolism of testosterone or epitestosterone 16a-OHTestosterone 27 13 catalyzed by cytochrome P-450b, suggesting that hydrogen 160-OH 28 9 peroxide, superoxide anion,or hydroxyl radicals were not 12 Androstenedione 25 15a-OH Epitestosterone 24 5 involved in the catalytic process. Neither hydrogen peroxide 4 16a-OH 22 (18 mM) nor cumene hydroperoxide (0.1 or 1.0 mM) in the 22 21 160-OH absence of NADPH was capable of converting testosterone or Androstenedione 20 35 epitestosterone to their respective 16a- or 16P-hydroxylation 17-Desoxytestosterone 16a-OH 0.3 17 products, or to androstenedione at any more than 3% of the 160-OH 0.3 112 rate of the NADPH-dependent monooxygenase reactions (data not shown). Also, the addition of a hydrogen peroxidePM) substrate concentration. With epitestosterone, 15a-, generating system consisting of glucose (2 mM) and glucose 16a-, and 16i3-hydroxylation and androstenedione formation oxidase (14 units/ml) to the standard incubation (complete had the same apparent K,, 20-24 p ~which , was not signifi- reaction) mixtures resulted in a decrease of approximately cantly different from the values obtained with the analogous 50% in 16-hydroxylation and androstenedione formation with reactions of testosterone. In comparison to testosterone, there either steroid substrate, presumably as a result of enzyme was a marked (5-fold) stereoselectivity in the (2-16 oxidation inactivation (data not shown). of epitestosterone in favor of &hydroxylation, and androKinetic Isotope Effects stenedione formation was enhanced both with respect to absolute turnover number and as a percent of total metaboIsotopic substitution of hydrogen can lead to a decrease in lism (Table I and Fig. 1). Furthermore, 15a-hydroxylation the rateof product formation if carbon-hydrogen bond break-
17327
Cytochrome P-450b-dependent Androgen Oxidation age is involved in a rate-limitingsurface of the reaction profile. To further characterize the mechanism of oxidation of testosterone and epitestosterone by cytochrome P-450b, the effect of isotopic substitution of hydrogen at C-16 and C-17 on the kinetic parameters associated with oxidation at these sites was determined. The results obtained from noncompetitive deuterium isotope effects associated with metabolism of [ 17-’H]- and [16,16-2H2]testosteroneand epitestosterone are shown in Table 11. Deutero and proteo substrates were all incubated at V,,, conditions. Rates of product formation were not adjusted to account for the fact that incorporation of deuterium into the substrates during synthesiswas not 100% (see “Experimental Procedures”).Deuterium incorporated in the 17-position decreased the rate of androstenedione formation 3.4- and 2.4-fold for testosterone and epitestosterone, respectively. When deuterium was located at the 17-carbon, little effect was seen on the rate of 16a- or 168-hydroxylation of testosterone, but a small increase was seen in the rate of 15- and 16-hydroxylation of epitestosterone,perhapsasa result of metabolic switching (19, 20). Substitution of deuterium at the16-carbon resulted in substantial decreases in rate of 16a- and168-hydroxylation for both steroids, data consistent with the hydrogen atom abstractionrecombination mechanism proposed for aliphatic hydroxylations catalyzed by cytochrome P-450 (21, 22). No significant decrease in rate was observed for oxidation at the 17-carbon with either substrate or in the rate of 15a-hydroxylation of epitestosterone. These results eliminate the involvement of a C-16 carbonhydrogen bond in a rate-limiting process associated with C15 or C-17 oxidations. Definitive proof that theC-16 carbonhydrogen bond is not involved in androstenedione formation was obtained by isolating the androstenedione formed from [16,16-2H2]testosterone and establishing, by mass spectral analysis, thatthere was essentially complete retention of deuterium in the metabolite (Fig. 2). Lineweaver-Burk double-reciprocal plots for androstenedione formation (Fig. 3) indicated that 16- or 17-deutero substitution of testosterone had no effect on the apparent K,,, of the reactions. Similarly, TABLEI1 Oxidative metabolismof testosterone, epitestosterone, and their deuterated analogues by a purified monooxygenase system reconstituted with cytochrome P-450b Reaction mixtures, reconstituted with 0.05 or 0.10 nmol of cytochrome P-450b for the turnover studies, were as described under “Experimental Procedures.” Time of incubation was for 5 or 10 min at 37 “C. Steroid derivative
[16,16-’H2]
18
2
Z W
0
W
a N z
2
c
V
z a
0
z
s
3
m U
> c
a
W
W V
a
t-
a N z
2
-
J
a
c
0
t-
-
C
J
Z
W
0
W
c
J
-
a
t0 t-
U
a 0 z
3
U
> c
-
m
J
W
v
a
Z W
-
V
a W a
a
-
1
W
n
250
3
m/z FIG. 2. Mass spectrum of androstenedione formed metabolically from incubation of testosterone ( A ) and [16,16-*H~]testosterone (B)with a purified monooxygenase system reconstituted with cytochrome P-450b (0.10nmol). Methylene chloride extracts of 10 replicate incubations werepooled prior to the isolation of the androstenedione. See “Experimental Procedures” for reaction conditions, extraction and isolation procedures, and conditions for the GC/MS analysis. m/z
I.0-
-
4 -I>
0.41
I
./J
/
/
Turnover no. Metabolite Testosterone Epitestosterone nrnol/rnin/nmolP-450
Unlabeled
100-
100
15a-OH 16a-OH 16P-OH Androstenedione Total
14 10 13 37
15a-OH 16a-OH (21)“ 3 16P-OH 2 (20) Androstenedione 16 (123) Total 21 (57) 15a-OH 16a-OH 13 (93) 16P-OH 10 (100) Androstenedione (29) 4 Total 27 (73)
6 5 44 59 114
5 (83) 0.6 (12) (14) 6 51 (86) 63 (55) [17-’H] 10 (167) 8 (160) 72 (164) 25 (42) 115 (101) ’Values in parentheses are the reaction rates expressed as a percentage of the analogous reaction rate observed with the parent steroid.
0.05
0.10
0.15
0.20
I S (pM) FIG. 3. Lineweaver-Burk double-reciprocal plot of rate of androstenedione formation as a function of the concentration of testosterone or two deuterated analogs of testosterone in a purified monooxygenase system reconstituted withcytochrome P-450b (0.04 nmol). Incubation time was for 3 min at 37 “C. Curve fitting was by an unweighted least squares regression analysis and r z values were 20.93.
no K,,, effects were seen for the C-16 oxidations (data not shown). Taken together, these data indicate that the C-17 carbonhydrogen bond is broken in a rate-limiting enzymatic step during cytochrome P-450b-catalyzed oxidation of both testosterone and epitestosterone to androstenedione.
17328
Cytochrome P-450b-dependent Oxidation Androgen @O-
Effect of Deuterated Water on the Metabolism of Testosterone and Epitestosterone The effect on metabolism of substituting deuterium for hydrogen on the 17-hydroxy groups of testosterone and epitestosterone, in 'H20, was investigated. The reactions with the deutero analogs were performed in 85% deuterium oxide to prevent exchange of the istope with water. A decreased rate of steroid metabolism at both the C-16 and c-17 positions of testosterone and epitestosterone was observed (data not shown), and this appears to represent another example of inhibition of the cytochrome P-450-dependent monooxygenase system (and certain otherenzyme systems) by deuterium oxide (23). However, the extentof inhibition of (2-16 hydroxylation was the same as inhibition of (2-17 oxidation for each steroid, suggesting that androstenedione formation from either substrate does not involve the breaking of the hydrogen-oxygen bond of the 17-hydroxylgroup in the rate-limiting step. Metabolite Formation in the Presence of 1802 The ability of cytochrome P-450b to catalyze the 17a- and 17P-hydroxylation of testosteroneand epitestosterone, respectively, was evaluated with incubations of the monooxygenase system in an atmosphere of "02.One postulated mechanism of androstenedione formation is through a gemdiol intermediate which can rearrange to thecarbonyl product with loss of water. This mechanism of C-17-hydroxylation would beexpected to yield approximately 50% "0 enrichment in the 17-keto group of androstenedione because the dehydration of a gem-diol intermediate should occur spontaneously with an equal probability of either oxygen being lost in the absence of steric or electronic effects. Comparisons of the mass spectra of the androstenedione formed from testosterone and epitestosterone in an atmosphere of " 0 2 show markedly different results (Fig. 4). Whereas net incorporation of "0 into androstenedione, when testosterone was the substrate, was only 4.8% (approximately twice background), the androstenedione formed from epitestosterone was 84% enriched in "0. These data are summarized in Table I11 (Experiment l ) , along with additional data which show that the low incorporation of "0 with testosterone was not due to its exchange with solvent water, because incubations with H2"0 did not lead to incorporation of "0 into androstenedione (Table 111, Experiment 2). Incubation of the [17-'80]androstenedione, formed from epitestosterone in an incubation system devoid of cytochrome P-450b, and reisolation of the androstenedione by HPLC also indicated that no exchange of oxygen took place during the preparation of the samples prior to mass spectral analysis. Incorporation of " 0 2 into the 16-hydroxylated products of both substrates was at theexpected levels of 100% in all experiments, demonstrating that 16-hydroxylations were true monooxygenase reactions and that the atmospheres in the reaction mixtures were completely equilibrated with 1 8 0 2 . Effect of Temperature on "0 Incorporation The marked difference in extent of "0 incorporation at C17 of testosterone and epitestosterone,relative to a 50% level of incorporation expected for a gem-diol mechanism, suggested that either cytochrome P-450b was stereoselectively directing the loss of water from the gem-diol or an alternative mechanism was operative. This hypothesis was approached experimentally by performing a series of reactions at different incubation temperatures in an attempt toalter the activation energy of the possible pathways to product. In particular, we postulated that elevated incubation temperatures could en-
-
100-
A.
- 55
287
B
207
-
50
-
z W
v
z
0
c a N z
-
B z 3
0
W
v
-
a 0 z
-
Z
m
H
W
I-
W
c c
I - -
-
W V
e
-
W
a
-> c a J W
e
J
a
O
-
Z
a a
z 0 c a N z
0
3
m
a
H
-
J
a
I-
> -
c
a
I-
J W
O
z W V
-
IL W
a
-
L
-
189
250
250
300
Ir
3
m/z
287
50
89
50
2
z
0 e
9 c
2
z
U
a N
3
s
s
-I
-I
U
a c 0 c
c c c
0
I-
z
z
V
0
W
W
a W a
(L
W
a
L289
250
m /z
Y
3 I
3 1 m12
FIG. 4. A comparison of the mass spectra of metabolically formed androstenedione from incubations of testosterone (upper panels) or epitestosterone (lower panels) with a purified monooxygenase system reconstituted with cytochrome P-450b and under either an ''02 atmosphere (panel A) or an ''02 atmosphere ( p a n e l B ) .
hance the dissociation of the proposed gem-diol intermediate from the cytochrome and might permit loss of water from either face of the steroid with equal fascility. Incubation of testosterone with the reconstituted monooxygenase system under an "02 atmosphere at 47 "C decreased the turnover number approximately 60% relative to the rate at37 "C and modestly, but significantly, increased "0 incorporation into androstenedione (Table 111, Experiment 3). Similarly, incubation of [ 17-'80]testosterone with the monooxygenase system in air at 47 uersus 37 "C resulted in a small, but significant, decrease in percent recovery of "0 in androstenedione (Fig. 5, Table IV), but notin 16a- or 16@-hydroxytestosterone (Table IV). However, the extent to which androstenedione was enriched in "0 when epitestosterone was the substrate remained essentially the same at incubation temperatures
17329
Cytochrome P-450b-dependent Androgen Oxidation TABLE I11 "0 incorporation into C-19 steroid metabolites formed by a purifiedmonooxygenuse system reconstituted with cytochrome P-450b Incubation conditions, metabolite analyses, and mass spectral determinations were as described under "Experimental Procedures." Substrate and incubation temperature
Atmosphere
Water
Metabolite
"0 incorpo. ration %
Experiment 1 Testosterone (37 "C) Testosterone (37 "C) Testosterone (37 "C) Epitestosterone (37 "C) Epitestosterone (37 "C) Experiment 2 Testosterone (37 "C) Testosterone (37 "C) Testosterone (37 "C) Experiment 3 Testosterone (47 "C) Epitestosterone (15 "C) Epitestosterone (47 "C)
'802
1802
Air Air Air '802 '802 '802
16a-OH 16P-OH Androstenedione 16P-OH Androstenedione
100 100 4.8 100 a4
16a-OH 16@-OH Androstenedione
0 0 0
Androstenedione Androstenedione Androstenedione
7.8 a4 a4
the formation of nominal amounts of 15a-hydroxyepitestosterone. Apparent K,,, values for the (2-16 and C-17 oxidations are comparable to those observed with testosterone (20-30 PM). 17-Desoxytestosterone is an exceptionally good substrate for cytochrome P-450b, exhibiting an apparent K , of 0.3 FM, which was almost 2 orders of magnitude lower than values observed with testosterone or epitestosterone. Oxidation ocDISCUSSION curs exclusively at C-16, with @-hydroxylationpredominating The present study was undertaken to further define the over a-hydroxylation by a ratio of almost 7 to 1. The V,,, for effect of steroid structure onstereoselective product formation 16@-hydroxylationof 17-desoxytestosterone is 12times higher catalyzed by cytochrome P-450b, and to elucidate the mech- than the rateobserved for the 16@-hydroxylationof testosteranism by which testosterone and epitestosterone areoxidized one. Ortiz de Montellano et al. (24) and Kunze et al. (25) have at C-17 to form androstenedione. With respect to the stereoselectivity of product formation, shown that the chiral orientation of the heme in cytochrome we previously reported that a purified monooxygenase system P-450b is identical to thatfound in hemoglobin and thatonly reconstituted with cytochrome P-450b metabolizes testoster- a single approach to the pyrrole A-ring exists. This, in conone exclusively in theD-ring at C-16 and C-17 (4). Oxidation junction with the D-ring selectivity of androst-4-en-3-one on the a- and @-faceat C-16 occurred with equal facility, i.e. oxidation by cytochrome P-450b, suggests that theactive site 16a- and 16@-hydroxytestosteronewere formed from testos- of the enzyme is a pocket of dimensions such that thesteroid terone in approximately equal molar amounts. Androstene- substrate can only bind in one catalytically competent condione was metabolized by the monooxygenase system recon- formation-that with the C-16 (the distal carbon) closest to stituted with cytochrome P-450b exclusively at the C-16 po- the heme and a preference for reaction on the @-faceof the sition, but the overall rate of metabolism wastwice that steroid. However, as the molar volume of a @-faceC-17 subobserved with testosterone andthe ratio of 16@-hydroxylation stituent increases (progesterone > testosterone > epitestosrelative to 16a-hydroxylation was greater than 1O:l. This terone > 17-desoxytestosterone), there is a shift to a-oxidamarked change in stereoselectivity in favor of the @-faceat tion, without an affect on apparent binding affinity until the C-16 resulted from an &fold enhancement in 16@-hydroxyl- substituents areremoved (17-desoxytestosterone). It has been ation and a 2-fold diminution in 16a-hydroxylation relative previously postulated that the active site of cytochrome Pto testosterone. The presence of a P-acetyl substituent at C- 450b is hydrophobic in nature (6). This observation is sup17 (progesterone) results exclusively in 16a-hydroxylation of ported here by the fact that desoxytestosterone has a much lower apparent K,,, than theother substrates. Thus,the active the steroid (6). In the present study we show that two additional steroids, site of cytochrome P-450b appears to be a hydrophobic cleft epitestosterone and 17-desoxytestosterone, are also metabo- above the pyrrole A-ring that binds the steroid in an orienlized by cytochrome P-450b exclusively in the D-ring, and tation perpendicular to theheme. Absence of a substituent at that the presence and orientation of the C-17 substituent C-17 is associated with a low apparent K,, high V,., exclusive markedly affects catalytic rates, apparent substrate affinity, metabolism at C-16, and a marked preference for @-oxidation. Presence of a keto or a-hydroxy moiety at C-17 preserves the andthe stereoselectivity of the reactions, as well as the apparent mechanism of oxidation. The metabolism of epites- preference for @-oxidationat C-16, whilea 17@-hydroxygroup tosterone is characterized by a marked stereoselectivity in or a 17@-acetylmoiety leads to a progressive shift in favor of favor of 16@-hydroxylation(4- to &fold relative to 16a-hy- 16a-hydroxylation. With respect to metabolism at C-17, it is droxylation), a rate of androstenedione formation that is three of interest that cytochrome P-450b readily catalyzes the oxito five times higher than thatobserved with testosterone, and dation of a 17-hydroxy to a 17-keto (with 17a-hydroxy oxifrom 15-47 "C (Table 111, Experiment 3). Although the temperature effects are small, the data indicate that fundamentally different processes are operating in the formation of androstenedione from testosterone and epitestosterone and provide important insights (discussed below) into the mechanism of the reactions.
17330
Cytochrome P-450b-dependent AndrogenOxidation
250
300 mh
250
300 mh
FIG. 5. Mass spectra of androstenedione formed metabolically from incubation of [17-'80]testosterone (55%)at 37 OC (top)or 47 O C (bottom)with a purified monooxygenase system reconstituted with cytochrome P-450b. See "Experimental Procedures" for reaction conditions, extraction and isolation procedures, and conditions for the GC/MS analysis.
TABLEIV " 0 retention in metabolites formed from [l 7-'80]testosterone incubated with a purifiedmonooxygenase system reconstituted with cytochrome P-450b The monooxygenase system was reconstituted with 0.10 nmol of cytochrome P-450b and incubated for 10 min at the indicated temperatures. Values represent the results from two separate experiments. Total rates of metabolism at 47 "C were 33 and 27% of the resuective rates at 37 "C for the two exueriments. Incubation temperature
[180]Testosterone" [lsO]Androstenedione
"C
%
37 47
55,49 53,50
%
16a-OH %
53,50 52,52 42,41 57,52 "The [17-'80]testosteronesubstratecontained 55% "0, as described under "Experimental Procedures." The values reported above for testosterone were determined from substrate isolated from the incubation mixtures.
dation occurring at a severalfold greater rate than l7P-hydroxy oxidation), but no oxidation occurs at C-17 of 17desoxytestosterone (Fig. 1) or progesterone (6). A major focus of the present study was to gain an understanding of the mechanism bywhich cytochrome P-450b catalyzes the oxidation of the C-17 hydroxyl to a keto. Two mechanisms of androstenedione formation not directly involving the cytochrome were readily eliminated. First, complete dependence of the reactions on cytochrome P-450b, NADPH (but not NADP or NAD), NADPH-cytochrome P450 reductase, and molecular oxygen,coupled with the potent and complete inhibitory effect of monoclonal antibodies to two distinct epitopes on cytochrome P-450b, demonstrate that the conversion of the 17-hydroxy function to a 17-keto function is not being catalyzed by a dehydrogenase or another contaminating enzyme. Secondly, scavengers of various active oxygen species did not inhibitproduct formation, nor was the reaction supported by peroxides, indicating a monooxygenase reaction was operative. We had previously discussed the possibility that cytochrome P-450b may indirectly catalyze formation of androstenedione via an initial oxidation of testosterone at the(2-16 position with intramolecular hydrogen transfer and subsequent dehydration of the hypothetical oxy intermediate to yield androstenedione, as well as the 16a-and 16P-hydroxylation products (4). Unequivocal evidence that thismechanism was not operative in the cytochrome P-450b-dependent oxidation of testosterone to androstenedione was obtained from the mass spectral analysis of the androstenedione formed which demonstrated acomplete from [ 16,16-2H2]te~to~ter~ne, retention of the two atoms of deuterium. Further evidence that theC-16 and C-17 oxidations are notcoupled comes from the observation that therate of 16-hydroxylation of testosterone is not affected by substitution of deuterium at C-17 and that the C-16 hydroxylation products formed from [17-'H] testosterone have retained the deuterium. The currently accepted mechanism for the direct hydroxylation of carbon-hydrogen bonds by cytochrome P-450 is a stepwise process involving the formation of a carbon-centered free radical generated by hydrogen abstraction via an activated oxygen,followedby rapid collapse of the resulting carbon radical-perferric hydroxide radical pair (21, 22, 26). The recombination is presumably a cage reaction with a low energy barrier and extremely high rate (21, 27) because most of the reactions investigated proceed with retention of stereochemical fidelity (28-31). If the recombination reaction was slow, stereochemical scrambling would beexpected. However, stereochemical scrambling is relatively slight, even with norbornane (32) and camphor (33), two chemicals whose metabolically induced scrambling has been most studied. For certain compounds, the recombination reaction involves reaction of the perferric hydroxide radical (effectively a hydroxyl radical) with a hydrogen attached to a carbon adjacent to the initial carbon radical to generate an alkene and water (34). The data for androstenedione formation reported here can be understood in terms of the above discussed mechanism for direct hydroxylation. The primary isotope effects observed indicate that theC-17 carbon-hydrogen bond is involved in a rate-limiting step in the formation of androstenedione from both testosterone andepitestosterone, implicating the resulting intermediate as the C-17 carbon radical (Fig. 6). Unlike traditional aliphatic oxidations, an oxygen atom instead of carbon is adjacent to the radical. Nevertheless, the same possibilities exist: first, oxygen recombination can occur to form a gem-diol (recombination mechanism), and/or second, abstraction of the hydrogen atom on the oxygen occurs to
Cytochrome P-450b-dependent AndrogenOxidation
I-".
17331
1
-H-
A n d m FIG. 6. Proposed mechanism for the cytochrome P-45Ob-dependent formation of androstenedione from testosterone and epitestosterone through gem-diol and dual hydrogen abstraction pathways. Dark arrows indicate preferred routes. Only the C and D rings of the steroids are shown. The asterisk denotes "0.
form a carbonyl (dual hydrogen abstraction mechanism). As one with rat hepatic microsomes under an "02atmosphere, gem-diols are in equilibration with carbonyls and the equilib- although no quantitative datawere presented. The high degree of retention of label following the metabrium is pushed to the directionof the carbonyl, thegem-diol would be expected to rearrange spontaneously with loss of olism of epitestosterone is strong evidence in support of a gem-diol mechanism whereby formation of the gel-diol interwater to give the carbonyl. Studies investigating aliphatic hydroxylation utilizing 1802 mediate is followed by directed loss of water from the a-face have shown that all oxygen the incorporated into the substrateof the steroidmolecule (Fig. 6). Several factors could contriboriginates from molecular oxygen (21, 22). However, a gem- ute to thestereoselective rearrangement of gem-diol to proddiol formed in this manner should theoretically retain only uct. The chiral orientation of the enzyme-substrate complex could be such thatgem-diol eliminates waterexclusively from 50% of the label if the molecule issymmetricalandrearrangement to a carbonyl is not governed by properties of the a-face with the assistance of a functional group in the the enzyme. The "02studies reported here show that only active site, or alternately, substrate structural features may 5% of the stable isotope was retained in androstenedionefrom direct theeffects. A structural feature on the steroid which may be involved testosterone while 84% was retainedupon metabolism of in the stereoselectivity of thereaction is the bulky C-18 epitestosteronetoandrostenedione.Controlexperiments of oxidation at C-17.4In the showed that the amount of isotope incorporated was not methyl group adjacent to the site gem-diol mechanism, the steric interactions would result in affected by solvent or atmosphere. These results for testoswater being lost from the least hindered side, the side which terone are clearly different than those reported by others. can most readily interact with a solvent water molecule. The Studies by Cheng and Schenkman(35) utilizing a monooxysolvent water molecule then plays the role of acidic or basic genase system containing purified cytochrome P-450 RLM5 catalyst to potentiate eliminationof water. Indirect evidence showing no discernible incorporation of "0 from " 0 2 into in supportof this hypothesis is that direct chemical reduction androstenedione formed from testosterone. These investiga- of androstenedione with various metals and metal hydrides tors found no evidence for oxygen exchange with water or in attempts to form epitestosterone (addition of hydride to atmospheric oxygen and concluded that androstenedione for- the P-face) alwaysresulted in>95% formation of testosterone mation catalyzed by cytochrome P-450 RLM5 proceeded via (addition of hydrogen to the a-face) in the pathways (data a "peroxidative mechanism." Using cytochrome P-450,,,11 pu- not shown). rified frompig testes microsomes, Suhara et al. (36) were unable to demonstrate incorporation of "0 from "02when We were able to obtain very small quantities of 18-nortestostertestosterone was metabolized toandrostenedione.Onthe one. Unfortunately, absence of the 18-methyl group markedly reduces other hand, Nakamura(37) has reported incorporationof "0 the solubility of the steroid, and we were unable to discern any into androstenedione formed from incubation of epitestoster- metabolism under a variety of incubation conditions.
17332
Cytochrome P-450b-dependent Androgen Oxidation
At first glance, the "0retention in testosterone would seem to support a gem-diol mechanism with stereoselective loss of water. However, if both testosterone and epitestosterone are oxidized to androstenedione through agem-diol pathway, the amount of isotope retained should be opposite (e.g. 40% for one, 60% for the other) since the intermediate gem-diol in both instances is the identical molecule. If the mechanism were identical for testosterone, one would expect 16% of the isotope to be retained. The fact that only 5% is retained suggests that an additional mechanism is operating for testosterone. Also supporting this view is the observation of a temperature effect on "0 incorporation into testosterone, but not epitestosterone, strongly suggesting that thedifference in "0 incorporation is governed by transition state differences from a partitioningof mechanisms. In a manneranalogous to the formation of alkenes in the biotransformation of alkanes (34), the additional mechanism (dual hydrogen abstraction) could involveinitial carbon radical formation at C-17, reaction of the perferric hydroxide radical with the hydrogen attached to the C-17 hydroxyl, and collapse of the resulting diradical to theketone. For the dual hydrogen abstraction reaction, no isotope would be incorporated with either substrate (Fig. 6). If the assumption is made that themetabolism of epitestosterone is completely through the gem-diol pathway, then the relative contribution of each of the two pathways to androstenedione formation from testosterone as well as the free energy difference between the two transition states can be approximated. Accordingly, the ratio of loss of water from the a- uersus the 6-face is 84/16 or 5.3 with a free-energy difference of approximately 1 kcal/mol. Thus, if 5% of the oxygen retained on testosterone is from a-retention of oxygen from the gem-diol, then 26% is lost from the gem-diol from the aface.Accordingly, 31% of the products must then arise through agem-diol mechanism and 69% by the dual hydrogen abstraction mechanism. Assuming that the ratio of products formed from each pathway is directly related to the relative equilibrium rate constants associated with these pathways, the transition state free energy difference between these two pathways in theformation of androstenedione from testosterone is approximately 0.5 kcal/mol. While the system described here is different from chemical experiments where recombination of a carbon radical uersus abstraction to form a carbonyl were measured (38,39),the transition statefree energies are of the same magnitude. To summarize, data on the oxidation of epitestosterone to androstenedione strongly suggests a gem-diol intermediate with subsequent substantial enzyme or substrate-directed dehydration. The data for testosterone does not eliminate a similar mechanism, but does suggest that another mechanism besidesgem-diol (dual hydrogen abstraction) may also be involved. Acknowledgments-We express our appreciation to the Steroid Reference Collection, Medical Research Council, London, for donating reference steroids, and toKaren Schreck and Cathy Michaud for expert preparation of the manuscript. REFERENCES 1. Hall, P. F. (1985) Vitam. Horm. 42, 315-368 2. Conney, A. H., Levin, W., Ikeda, M., Kuntzman, R., Cooper, D. Y., and Rosenthal, 0. (1968) J. Biol. Chem. 243,3912-3915 3. Conney, A. H., and Kuntzman, R. (1971) in Concepts in Biochemical Pharmacology, Handbook of Experimental Pharmacology Series (Brodie, B. B., and Gillette, J., eds) pp. 401-421, Springer-Verlag, Berlin
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