Synthesis of 2-Bromoacetamidoestrone Methyl Ether and Study

THE JOURNAL OF BIOLOGICAL CHEMISTRY

Prrnted m U.S.A. Vol. 255,. No. 8, Issue of April 25. pp. 3660-3664. 1980

Synthesis of 2-Bromoacetamidoestrone Methyl Ether and Study of the Steroid-binding Site of Human Placental Estradiol 17/3-Dehydrogenase*

(Received for publication, September 4, 1979, and in revised form, January 14, 1980)

Chang-Chen Chin, Pierre Asmar, and James C. Warren From the Department of Obstetrics and Gynecology and the Department of Biological Chemistry, Washington University School of Medicine, St. Louis, Missouri 63110

To characterize further the active site of human placental estradiol 17P-dehydrogenase (EC 1.1.1.62), we have synthesized 2-bromoacetamidoestrone methyl ether. The affinity-labeling steroid is a substrate for the homogeneous enzyme. It inactivates the enzyme in a time-dependent, irreversible manner which follows pseudo-first order kinetics. Further, inactivation conducted with varying steroid concentration displays saturation kinetics. When 1.7 X lo-‘ M enzyme is inactivated by 2.6 X M 2-bromoacetamidoestrone methyl ether, the presence of an equimolar concentration of estradiol or 5.2 X M concentrations of NAD+, NADP+, or NADPH markedly slow the rate of inactivation. Bromoacetate (2.6 X M) does not inactivate the enzyme.

After inactivation with 2-brom0[2’-’~C]acetamidoestrone methyl ether, amino acid analysis reveals carbox- methylated derivatives of cysteine, histidine, and lysine containing 65,25, and 88, respectively, of the total incorporated carboxymethyl groups. The presence of estradiol, NADP+, or NADPH clearly inhibits alkylation of cysteinyl and histidyl residues and slows the rate of enzyme inactivation. Protection of these residues by both estradiol and NADPH suggests that they may actually be in the cofactor region of the active site, that this region is close to the steroid A-ring, and that binding of cofactor, by physical interposition, denies the reagent-bearing steroid access to these residues.

Estradiol 17P-dehydrogenase (EC 1.1.1.62) has been iso- lated from human placenta in homogeneous form by affinity chromatography in this laboratory (1) and by Nicolas et al. (2).

Subsequently, we crystallized this enzyme (3) and studied the topography of the steroid-binding site with affinity-labeling techniques using 16a-bromoacetoxyestradiol 3-methyl ether, which revealed a histidyl residue present in the catalytic region of the active site (4), and 4-bromoacetamidoestrone methyl ether, which revealed the presence of cysteinyl and lysyl residues proximating position 4 of the bound steroid (5).

Similar studies of another enzyme, the 2OP-hydroxysteroid dehydrogenase (EC 1.1.1.53) from Streptomyces hydrogenans, have been previously carried out in this laboratory with affinity-labeling techniques. In that case, 6P-bromoprogester- one and 6/3-bromoacetoxyprogesterone alkylated an enzyme cysteinyl residue proximating steroid position 6 (6, 7); 2a-

* This research was supported by Research Grant AM-15708 from the National Institutes of Health. 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 accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

bromoacetoxyprogesterone and lla-bromoacetoxyprogesterone alkylated different methionyl residues proximating positions 2 and 11 (7,8); and cortisone 21-iodoacetate (9) and 16a- bromoacetoxyprogesterone (10) alkylated a hisidyl residue present in the catalytic region of the active site. These results led us to conclude that an affinity-labeling steroid will not only identify amino acid residues at the steroid-binding site, but will even identify those that proximate the reagent-bearing region of the steroid derivative as it undergoes the reversible binding step. Therefore, affinity-labeling methodology should allow evaluation of the comparative topography of the steroid-binding sites of various proteins.

In the present study, we have synthesized Z-bromoacetamidoestrone methyl ether and used it to affinity-label human placental estradiol 17/3-dehydrogenase. Evidence will be presented that this enzyme has cysteinyl and histidyl residues which proximate the upper A-ring of the steroid as it binds at the active site.

EXPERIMENTAL PROCEDURES’

RESULTS

Inactiuation of Estradiol 17/3-Dehydrogenase by 2-Bro- moacetamidoestrone Methyl Ether-The inactivation of estradiol 17fi-dehydrogenase by 2-bromoacetamidoestrone methyl ether was carried out in Buffer A containing 20% ethanol (v/v) because of the low solubility of this steroid in aqueous solution. When 2.57 m o l (180 pg) of estradiol 17P- dehydrogenase was incubated with 0.39 pmol (150-fold molar ratio) of 2-bromoacetamidoestrone methyl ether in 1.5 ml of Buffer A containing 20% ethanol, pH 7.0, 25OC, the inactivation (as determined by withdrawal of 50-4 aliquots and assay for remaining activity as described under “Methods”’) fol- lowed pseudo-first order kinetics with half-time, tlp, of 10 h (Fig. 3). In the presence of 0.39 pmol of estrone or estradiol, the inactivation is slowed because both of these steroids are competing for the active site. In the presence of 0.77 pmol of NAD”, NADP+, or NADPH, the inactivation was even more inhibited. During a 24-h period, the control enzyme sample retained its original activity.

Identification of Amino Acid Residues AZkyZated by 2- Bromo[Z’-’“ CJacetamidoestrone Methyl Ether-Enzyme was prepared as described above, except that the final enzyme concentration was M. Steroids and cofactors were added so that separate vials contained: ( a ) 1.5 X M 2-bromo[2’- 14C]acetamidoestrone methyl ether, (b) 1.5 X M 2-

I Portions of this paper (including “Experimental Procedures,” part of “Results,” Figs. 1, 2, 4, 5, and 6, and Refs. 11-17) are presented in miniprint immediately following this paper. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, Md. 20014. Request Document No. 79M-1810, cite author(s), and include a check or money order for $1.80 per set of photocopies.

3660

2-Bromoacetamidoestrone Methyl Ether

a0

30 0 4 a 12 20 24

TIME (Hours)

FIG. 3. Enzyme inactivation. Inactivation of 1.7 X 1O-6 M estra- dial l’i;C-dehydrogenase by 2.6 x 10m4 M 2-bromoacetamidoestrone methyl ether (B), 2.6 x 10e4 M 2-bromoacetamidoestrone methyl ether and 2.6 x 10e4 M estradiol (A), 2.6 X 10m4 M 2-bromoacetsrni- doestrone methyl ether and 5.2 x lo-” M NAD+ (0), 2.6 x 10m4 M 2- bromoacetamidoestrone methyl ether and 5.2 x 10d4 M NADP’ (O), 2.6 x 1O-4 M 2-bromoacetsmidoestrone methyl ether and 5.2 X 10m4 M NADPH (A), and the control or with 2.6 x lo-’ M bromoacetate (Cl). Each point is the mean of three determinations.

bromo[2’-“‘C]acetamidoestrone methyl ether and 3 X 10e3 M

estradiol, (c) 1.5 X 10m3 M 2-bromo[2’-‘4C]acetamidoestrone methyl ether and 1.5 X 10e3 M NADP+, and (d) 1.5 X 10m3 M

2-bromo[2’-‘4C]acetamidoestrone methyl ether and 1.5 X 10m3 M NADPH. After 14 h of incubation, 2-mercaptoethanol was added and the solutions were separately dialyzed and lyophilized as described above. Dry powders were dissolved in double-distilled water.

The remaining aqueous protein solutions (after protein determination and radioactivity quantitation) were separately lyophilized, then hydrolyzed with 6 N HCI at 110°C for 24 h; 98% of total radioactivity was recovered after acid hydrolysis The hydrolysis were evaporated to dryness in vacua and the residues were dissolved in 0.3 ml of 0.2 M sodium citrate buffer, pH 2.2. Aliquots (10 to 25 ~1) of the solutions were counted for total radioactivity and 0.25~ml samples were applied to an amino acid analyzer. The effluent fractions were collected every 30 s (0.4 ml) and counted in a scintillation spectrometer. A standard mixture of the carboxymethyl derivatives of cysteine, lysine, hiatidine, and other standard ammo acids were also run. Over 90% of the radioactivity applied to the column was recovered in all cases.

The enzyme sample that was incubated with 1.5 x lop3 M

2-bromo[2’-‘4C]acetamidoestrone methyl ether alone (67% inactivated in 14 h) displayed a peak corresponding in mobility to S-carboxymethylcysteine, which contained 65% of the total radioactivity. Peaks corresponding to the three carboxy- methylhistidines contained 25% of the total radioactivity. A small amount of radioactivity (8%) was present in peaks corresponding to the carboxymethyllysines.

Because of concern about high alcohol and steroid concentrations, it seemed important to repeat this experiment with 10m6 M enzyme concentrations using: (a) 5 X 10m5 M Z- bromo[2’-“Clacetamidoestrone methyl ether, (b) 5 X 10M5 M 2-bromo[2’-14C]acetamidoestrone and 10m4 M estradiol, (c) 5 X 10-s M 2-bromo[2’-‘4C]acetamidoestrone methyl ether and 5 X low5 M NADP+, and (d) 5 x 10m5 M 2-bromo[2’-‘*Cl- acetamidoestrone methyl ether and 5 x 10e5 M NADPH. Other than using larger volumes (so as to provide enough

enzyme to obtain detectable incorporation), lowering steroid concentrations (which allowed alcohol concentration to be reduced to lo%), and allowing the inactivation to proceed for 20 h, conditions and methodology of this experiment were similar to the one above. Again, in the aample with 2-bromo[2’- “C]acetamidoestrone methyl ether alone (43% inactivated in 20 h), S-carboxymethylcysteine contained 65% of the incorporated activity, peaks corresponding to the 3 carboxy- methylhiatidines contained 20%, and 7.7% was present in peaks corresponding to the carboxymethyllysines.

The effects of estradiol, NADP+, and NADPH on the alkylation of cysteinyl and hiatidyl residues by 2-bromo[2’- “C]acetamidoestrone methyl ether in both experiments are seen in Table I. It will be noted that alkylation is diminished in all cases, as is the extent of enzyme inactivation. Thus, estradiol, NADP+, and NADPH all inhibit alkylation of both cysteinyl and histidyl residues. In terms of lysine carboxy- methylation, the matter is different. The presence of e&radio1 does not inhibit; rather, it actually seems to increase the dicarboxymethyllysine content and has no effect on the quan- tity of monocarboxymethyllysine. NADP+ has no significant effect and NADPH reduces the content of both derivatives slightly.

DISCUSSION

The evidence indicates that 2-bromoacetamidoestrone methyl ether alkylates residues at the enzyme active site. The steroid is a substrate; the rate of enzyme inactivation follows saturation kinetics, indicating that the reversible binding step limits the irreversible alkylation step; inactivation of the enzyme is slowed by estradiol, which competes for the active site. Finally, inactivation parallels alkylation.

Inhibition of the alkylation of cysteinyl and histidyl residues by e&radio1 supports the concept that their alkylation does indeed occur by an affinity-labeling mechanism. The observed inhibition is to be expected in the case of NADP+, since Betz (18) has reported that the enzyme does not form an abortive ternary complex. Inhibition of alkylation by NADPH indicates that when cofactor binds, steroid has leas access to these residues, suggesting that they are actually in the cofactor- binding region of the active site and that this region is close to the steroid A-ring.

Steroids bearing alkylating reagent groups at positions 2,3, and 4 have now all been studied. If one examines Dreiding models of the various steroids used, certain conclusions can be drawn. A hiatidyl residue is alkylated by 2-bromoacetami-

TABLE I

Effect of estradiol and cofactor on the alkylation of specific amino acid residues of human placental estradiolli51(-dehydrogenase by

2-bromo[2’-“Clacetamidoestrone methyl ether (2-BAE) In the first experiment (data without parentheses): enzyme = 10e5

M, 2-BAE = 1.5 x 1O-3 M, estradiol (Ez) = 3 x 1O-3 M, NADP+ or NADPH = 1.5 X 10e3 M, inactivation time, 14 h. In the second experiment (data in parentheses): enzyme = 10m6 I, P-BAE = 5 x 10m5 M, estradiol = 10m4 M, NADP’ or NADPH = 5 x 1O-5 M, inactivation time, 20 h.

Steroids and cofac- Content of radioactivity in specific carboxy-

tar present during Inactiva- metbylated amino acids

the inactivation pe- tion riod DCM”- l-CM-His- 304&is- CM:Cys-

Hiitidine tidine telm?

0 dpm in thousands 2-BAE 67 (43) 18.1 (3.7) 27.1 (5.4) 31.5 (7.5) 201.5 (52.6) 2-BAE, Ez 42 (27) 7.0 (2.1) 10.3 (3.4) 18.2 (4.4) 56.6 (22.8) 2-BAE, NADP’ 24 (23) 9.9 (2.1) 11.8 (3.4) 17.3 (4.6) 42.6 (18.4) 2-BAE, NADPH 12 (15) 2.6 (1.2) 5.9 (2.0) 5.2 (2.6) 20.0 (9.6)

’ DCM, dicarboxymethyl.

3662 2-Bromoacetamidoestrone Methyl Ether

doestrone methyl ether and 3-iodoacetoxyestrone (19,20), but not by 4-bromoacetamidoestrone methyl ether (5). Thus, it must be spatially located (assuming that both derivatives are alkylating the same histidine) in the upper left region of the A-ring. With both 2-bromoacetamidoestrone methyl ether and 3-iodoacetoxyestrone (19,20), affinity labeling of histidine is inhibited by estradiol, as would be expected. With both derivatives, NADP’ also slows histidine alkylation, as would be expected, since the enzyme does not form an abortive ternary complex (18). NADPH does not inhibit histidine alkylation by 3-iodoacetoxyestrone (19,20), while with 24x0- moacetamidoestrone methyl ether it clearly does. While this inhibition of alkylation by reduced (evolutive) cofactor could occur via a change in enzyme conformation or affinity for the steroid, it is attractive to consider that it results from direct interposition of some portion of the cofactor molecule between the enzyme histidyl residue and steroid position 2. If so, it is reasonable to presume that the adenine moiety is involved, as one would expect the pyridine moiety (considering the stereo- specificity of hydrogen transfer by the enzyme) to proximate the steroid D-ring.

Cysteinyl residues are affinity-labeled by both 2- and 4- bromoacetamidoestrone methyl ether and 3-iodoacetoxyestrone. In all cases, estradiol, NADP’, and NADPH inhibit cysteinyl alkylation. These observations are also compatible with an interposition of some moiety of the cofactor between cysteinyl residues (which probably lie in the cofactor binding site) and the reagent groups on the steroid A-ring. Pons et al. (21) have shown by peptide analysis of the enzyme that 3- iodoacetoxyestrone affinity-labels 2 different cysteinyl residues, which are found in what they describe as chymotryptic Peptides C, and C5. These residues were alkylated about equally and the presence of reduced cofactor inhibited the alkylation of both. We presume that to be the case with 2- bromoacetamidoestrone methyl ether. But, since the enzyme is not susceptible to enzymic hydrolysis after alkylation with a bromoacetamidoestrone (22), we cannot be sure.

The inherent reactivity of the cysteine at pH 7.0 (as shown by reacting bromoacetoxysteroids with cysteine, tryptophan, histidine, lysine, and methionine) is at least 100 times greater than the other amino acids. Thus, should any “wallowing” of the steroid in the active site or “wrong way binding” occur, it would seem most likely that the artifactual alkylation result- ing would be of cysteine. Estradiol 17p-dehydrogenase con- tains 6 cysteinyl residues in each of its two, probably identical, subunits. Three cysteinyls (one in each of the chymotryptic Peptides C,, C3, and C,) on each subunit can be titrated by Ellman’s reagent and are prbtected by NADP+ (22). 3-Iodoac- etoxyestrone (reagent group on the A-ring) alkylates the cysteinyl residue in Peptides Cl and Cs about equally. While 3- chloroacetylpyridine-adenine dinucleotide (reagent group in a position which proximates the D-ring of the steroid as both bind at the active site) (23) affinity-labels the cysteinyl resi-

dues in all three peptides, the predominant labeling is of the residue in Peptide C3. We conclude that the cysteinyl residues in Peptides CI and C5 proximate the A-ring region of the steroid, that the cysteinyl in Peptide C3 proximates the D-ring region, and that all are shielded by cofactor. Nevertheless, the overlap noted with these two affinity-labeling compounds and the chemical reactivity of the cysteinyl residues of estradiol l7p-dehydrogenase, as compared to 20/3-hydroxysteroid dehydrogenase (24), leads one to conclude that the regional specificity of affiiity labeling observed in the case of 20p- hydroxysteroid dehydrogenase (4, 7, 8, 10) may not be fully applicable to the cysteinyl residues of the human placental enzyme.

A significant observation of this work is the clear suggestion that the adenine moiety of the cofactor proximates the steroid A-ring as they bind at the enzyme active site. Indeed, in those incubations containing reduced cofactor, we have routinely noted a radioactive peak on the amino acid analyzer which does not correspond to any known carboxymethyl amino acid. Work is now under way to evaluate possible affinity labeling of the adenine amino group of NADPH by 2-bromoacetamidoestrone methyl ether.

REFERENCES 1. Chin, C.-C., and Warren, J. C. (1963) Steroids 22,373-378 2. Nicolas, J. C., Pons, M., Descomps, B., and Crastes de Paulet, A.

3. Chin, C., Dence, J. B., and Warren, J. C. (1976) J. Biol. Chem.

4. Chin, C.-C., and Warren, J C. (1975) J. Biol. Chem. 250, 7682-

5. Bhatnagar, Y. M., Chin, C.-C., and Warren, J. C. (1978) J. Biol.

6. Chin, C.-C., and Warren, J. C. (1972) Biochemistry 11,2720-2725 7. Arias, F., Sweet, F., and Warren, J. C. (1973) J. Biol. Chem. 248,

8. Strickler, R. C., Sweet, F., and Warren, J. C. (1975) J. Bwl. Chem.

9. Ganguly, M., and Warren, J. C. (1971) J. Biol. Chem. 246,3646-

10. Sweet, F., Arias, F., and Warren, J. C. (1972) J. Biol. Chem. 247,

18. Betz, G. (1971) J. Biol. Chem. 246,2063-2068 19. Pons, M., Nicolas, J . C., Boussioux, A. M., Descomps, B., and

Crastes de Paulet, A. (1973) FEBS Lett. 36,23-26 20. Boussioux, A. M., Pons, M., Nicolas, J. C., Descomps, B., and

Crastes de Paulet, A. (1973) FEBS Lett. 36.27-30 21. Pons, M., Nicolas, J. C., Boussioux, A. M., Descomps, B., and

Crastes de Paulet, A. (1977) J. Steroid Biochem. 8,345-358 22. Pons, M., Nicolas, J. C., Boussioux, A. M., Descomps, B., and

Crastes de Paulet, A. (1976) Eur. J. Biochem. 68,385-394 23. Betz, G. (1968) Doctoral dissertation, University of Kansas Med-

ical School, Kansas City, Kans. 24. Bielmann, J. F., Braulant, G., Nicolas, J. C., Pons, M., Descomps,

B., and Crastes de Paulet, A. (1976) Eur. J. Biochem. 63,477- 481

Additional references may be found on p. 3664.

(1972) FEBS Lett. 23, 175-179

251,3700-3705

7686

Chem. 253,811-815

5641-5647

250,7656-7662

3652

3424-3433

2-Bromoaceta.midoestrone Methyl Ether 3663

Synthesis O f Z-BTanOdLetdmldDeltmne Methyl Ether and Study o f t he S te ra ld B lnd lng S l te Of Human P l a c e n t a l E r t r a d w l 178-Dehydmgenase

a f f l n l t y chmmdtography as repor ted frm t h l s l a b r a t o r y ( I ) . The f i n a l enzyme had d I P e C l f l C d c t l v i t y o f 1.1 umt r /mg and was bnageneous on d isc gel e l e c t m p h a r e n s a t pH 7.0 and 8.3.

Enzyme AIS~YS - Er t rad7o l 178-dehydrogenase was prepared fran hmdn placenta by

m l x l n g 0.3 ml o f 1 M NaHCO INa Cog buffer. pH 9 . 2 . 0.3 m o l O f es t rad io l -17a ~n 0.1 m l of ACt lY l ty was assayed according to Ldnger and Engel 113). The l l S d y was Conducted by

propylene glycol . 071 ml o i 251 hunan albumin, 1.0 ml o f 0.6 mM NAD+ and 1.45 m l of water

enzyme i n 50 ~1 Of buffer A (0.01 patarslum phosphate, 0.005 M EDTA and 201 g lycero l . t o a f l n a l v o l m e o f 2.95 ml. The r e a c t i o n was I n i t i a t e d by t h e a d d i t l o n O f 5-10 of

pH 7.0) k r a y r were c a r n e d a u t a t 2 5 O 5 1 i n I ~ e c k m n DU r E c t m p h m e t e r equipped with 1 G l l f o r d o p t i c a l c o n v e r t e r and 1 Coleman b d e l 165 Hitachi-Perkln-Elmer recorder. Enzyme a c t i r l t y was determined frm t h e i n i t i a l l lnear increase i n absorbance a t 340 m.

2-yl Brmoacetamide) - The synthesis of 2-bromaacetami&ertmne m t h y l e t h e r l n w l v e s four steps 11 n i t r a t i o n o f e s t m n e t o 2 - n i t r o e r t r o n e . 2 ) conver~lon o f 2 - m t r o e r t r o n e t o

methyl ether. and 4) r yn the r r r o f 2 -bmaace tam~doer t rone me thy l e the r (F igu re 1 ) . 2-n l t roe r tmne me thy l e the r . 3) reduc t ion Of 2 -n l tmer tmne methy l e ther to 2 -amlnoes tmne

Synthesjs of 2-Blwmacetamldoestmne Methyl Eth~~~3-methoxy-lI-oxo-1,3,5(IO]-ertratrien-

h g ; 3480 im-l and 1460 cm-l (-NH21. 1750 cm-l (17-C=Oj

4) Synthesis O f 2-0rmacetamldOertmne Methyl Ether (3-methoxy-l7-oxo-l.1,5~10)- e r t r a t r i e n - 2 - y l b r o m a c e t a m d e ) - To a s o l u t i o n o f 270 mg (0.8 m n o l ] o f 2-amno- estrone methyl ether i n 36 m1 O f dry methy lene ch londe. 100 mg ( 2 mol ) O f broma- a c e t i c acid and 600 mg ( 2 -1) of dlcyclohexylcarbodlimide. each ~n 12 ml of dry methylene chlonde. vere added ~ e w e n c e . The r e a c t i o n was c a r r i e d Out a t 0' fo r

dry&rr. i h e r e s i d u e was ex t rac ted w i th 10 ml O f acetone. The e x t r a c t was

The p l a t e r vere developed with chloroformlacetone (95.5) The malor U l t T a v l o l e t concentrated to 3-4 ml and chmmatographed on several 0 5 m r i l l c a g e l p l a t e s

ab io rb lng band5 were renowed f m the p la tes and ext racted wI th hot acetone The e x t r a c t # d l evaporated t o dryness under reduced pressure. and t h e H h l t l s h r e r l d u e ydl c r y s t a l l i z e d t w c e from ethanol to g ive 118 mg (31% y l e l d ) O f 1 1 h t y e l l o w c r y t a l s O f pure 2-bnmmacetaml OeStmne methyl e t h r r : 160-162m, A,#: 3250 m-l(NH ~n-NHCDtHp'drl, 1750 m-? (17-C.0). 1670 Cm- ( C = O In-NHCOCH28rj.

CZIH~~QJNBI.

Calculated C . 60.00, H : 6.19. N 3.33. BI: 19 04 Found: C - 59.10, H : 6 18. N 3.22, B Y : 18 76

1,3,5710)-estradtr~en-Z-~lbmul~2'-~~C]acetam~del - To d s o l u t i o n o f 7 5 mg (25 umalT of 2-aminoel t rone methy l e the in 0.5 ml of dry methy lene ch londe were added ~n sequence 0.67 mg (4 .9 mo l ) o f [ 2 ' - r4C lb romoace t l c ac ld (SpeC l f lC ac t i v i t y . 51 mCi/mcal), 2.04 mg

di~y~lohexylcarDo4llmide in 1 ml of d r y mthy lene ch londe . The r e d c t l m was Cdrrted a u t (14.7 ~ 1 ) o f b m o a c e t l c acid I n 1 ml of dry methy lene ch lor ide and 6.2 mg (30 mol) of

as described above f o r t h e u n l a b e l e d compound. After TeEryYStalI izat lm from ethanol. the Z-bmrm[2'-l4C]acetamidaestmne e t h y l ether r h m e d d s ing le spa t and a s i n g l e r a d l o a c t l w peak on t h i n l a y e r C h m d t o g m p h y w i t h a n Rf value I d e n t i c a l t o t h e u n l a b e l e d Cornpound y i e l d : 2.05 mg (2511. I p e C I f i c a c t l v l t y - 13.9 m C l I m m l , m.p.: 163-165°.

Synther r r O f Rad leac t l ve 2 -B rmac~ tamldoer t rone Me thy l E the r ( l -me thoxy - l l - axo -

RESULTS

Evidence that 2-Bmmoacetamidoeltrone Methyl Ether i s a Substrate for the Enzyme - The aszay was conducted by mix?ng estrone or 2-brormacetmidoestmne methyl ether ~n 0.1 rnl of propylene glycol , 0.3 ml of 1 y Potas57m pbsphate, pH 5.8, 0 1 ml of 25% hmdn albumln, 1.0 ml O f 0.3 NADH and 1.45 ml of w t e r t o a f i n a l volme o f 2.95 ml The reac t lon was i n i t m t e d by t h e a d d i t i o n of 10-20 Yg o f enzpne 7 " 50 u1 of buf fer A. Assays were c a r r i e d Out a t 25' f 1- and e n Z W a c t i v i t y Y I P detemlned frm t h e l n l t l d l l l n e a r decrease ~n absorbance a t 340 m. By va ry ing t he dmun t of Substrate fOT each assay and p l o t t l n g 11" VI. 118 by the method Of L i n w a v e r and BuI'k (15). 2-bmmaacetamldoertmne methyl ether has an appdm'ent & value of $3 x M and a Vmax value Of 2.03 nnal/m?n/ug enzyme (Figure 21 as c m p r e d yl 4.0 x 10- M and l.On n n l l m i n l v g enzyme respec t ive ly , fov e r tmne. Th ls indicates that 2-bmacetZmldooestmne methyl ether i s a SUbstPate f o r e s t r a d l o l 17fl- dehydmgenare and thus, must b ind a t tk a c t i v e r i t e .

-

I -10

Figure 2 L inA l raver a d Burk p l o t o f t h e reduction O f 2-brormacetanidoertrone methyl

Of buffer A. Each point Pepresents the mean o f t h ree a r rays . e ther by e s t r a d i o l 178-dehydrogenase; 9.8 uri ( e ) , and 19.6 ug ( 0 ) . ~n 0.05 ml

e r t m m f f w A before K i w t i c S t u d i e s o f t h e I n a C t i V d t l o n of Est rad io l 17f l -khydroqenare by 2-Bronoacetamldo-

inact ivat ion exper iments *?-e star ted . To each o f 8 tubes containing 0.66 nrm1 o f e r t r a d l o l

Z - b m a c e t m i d 0 e s t r a n e m t h y l ether d ls r0 lved ~n 0.14 ml of e t i ano l mak;ng d f lna l e thano l 178-dehydmgemse i n 1.1 n l o f b u f f e r A and 10% e thano l a t pH 7 25' * 1' was added

Concentrat ion O f 20%. The d ' f f e r e n t I t e m l d f i n a l C o n c e n t r a t i o n s used weye 0.45, 0.57, 0.85, 1.13, 1.69. 3.39 x lo-' M. These Incubations e r e cont inued for about 24 h r . Durlng

one 01 t v o - h r i n t e r v a l s frm each tube and arrayed fov remaining enzyme d c t l v l t y . I n the C O Y V ~ C O f t h e i n a c t i v a t i o n e r p e r l n e n t . 50 u1 of the Incubat,on mixture w d l Wthdrawn a t

add i t lon , a sample conta in ing no I t e m l d was used a s C o n t m l .

3664 2-Bromoacetamidoestrone Methyl Ether

i i g u r e 5 P l o t o f half time (g) inact ivat ion of est rad io l l t~-dehydmgenare by 2 - b m - acetamrdaertrone methyl ether ogainst the reciprocal of inactivatov concentration (111).

acetamideertmre Hethy1 Ether - To 2.07 w 130.4 n m l l of estradiol 178-dehydmgenare i n S t o i c h i m t r y o f I n a c t i v a t i o n o f E s t r a d i o l 17g-Dehydrnrrenase by Z - B ~ I ~ ~ [ ~ ' - ~ ~ C I -

9 rnl of hu f fe r A . 1 m1 O f ethanol was added. The solut ion was keot ovemioht a t 7- tem7perai;Fe: The next mming. th; en&! ranpie !&divided i n ' t a f raci ionr. Fract ion

i;;:i ~ ~ , " ; , " ~ ~ , ~ ~ : ~ ~ ~ ~ ~ z ~ ~ ~ w a ~ h ~ ~ c ~ ~ ~ ~ ~ ~ ~ ~ h ' ~ ~ ~ ~ ~ a ( ~ o ~ ~ ~ ~ 1 ) F ~ ~ ~ ! ~ ~ ~ ~ ~ . 1 4 ~ ] . 1. shich contained one-tenth of the enzyme vas mired wi th 0.1 ml o f e t h a n o l t o m i l e t h e

acetamldaertmne methyl ether dissolved i n 1 m1 o f e t h a m l m i r i n g t h e f i n a l e t h a m l concentrat lo" to 20%. Enzylne a c t i v i t y was assayed every 1 OP 2 hr.

acetamldaertmne methyl ether. 4.5, 4.5 and 1.0-ml povtions. respectively, of the reaction expemmnt. For the en?- sample (Fraction 27 undergoing inactivation with 2-bmmL2"

mixture were rermved at the t ime *hen 15, 48 and 75% inact ivat ion had occurred. They were nixed wi th 0.15 m l , 0.45 ml and 0.1 m l . rerpectively, O f 1.4 E 2-mrcaptoethaml to stop fu l lher Inac t iva t ion .

The contml (Fract ion 1) re ta ined i t s o r i ind l ac t i v i t y thmughout the course of thy

The three mercaptoethanol-treated enzym solutions then were separately dialyzed a g a l n s t d l r t l l l e d Water with frequent changer af the dialysate. Yhen the dialysate * I S found to contain radioactivity equal to backgmund. d i a l y s i s was terminated. The enzpne solutions then were l yophi l ized and me-tenth of the dry residue for each o f the enzpe SolUtlOnl were used for protein determination according to the method O f LoWY et a.116). The remin ing d ry powders then were dissolved i n 10 ml, 10 m l and 1 m l O f double d i s t i l l e d watel. respectively. 0.1 ml o f each so lut ion -as u d for quant i ta t ion O f rad ioact iv i ty . The resul ts ind lcate that 2.0 1.9 and 2.0 mol of [%]caraOxynethyl Qmupr incorporate for each r o l e O f Inact ivated en& a t 15. 48 and 75% inact ivat ion. respect ive ly LFigure 6).

3

An a l i q w t O f the radioactive enzyme hydmlyrate was run thmugh the amino acid analyzer C ~ - ~ r ( c c a l l i z ~ t i o n o f Rod>oactive Enzyme Hydmlysate wi th S-CarMXymethYlLysteine -

wi th no ninhydrin. The radioactive fractions corresponding to where standard S-carbaxy- wthy lcy r te ine shou ld e lu te were pooled. To t h i s f rac t i on w s added 1W mg Of standard

vas quant i ta ted w i th t r ip l i ca te a l i .awt l by sc in t i l l a t ion count ing . Another al iquot (1 ml) s-carboxmthy lcyr te ine and enough !dater to adjust the f inal volune t o 10 m1. Radioact iv i ty

W L treated with ninhydrin according to T r o l l and Cannon (17) to deternine the amino acid

u t i l m m l . The remaining solution was al lowed to Crystal l ize at 4'. The Crystals were content. The d s a y s were done i n t r i p l i c a t e and t h e s p e c i f i c d t t i w t y was Calculated I S

col lected by centr i fugat ion and FediPSOlved I n 1 a1 of water. The concentration of the resu l t i ng so lu t i im was assayed Ni th n inhydr in and Padloact iv i ty MI counted as described

MI 26.1, 21.1, 23.1 and 24.5 u C i / m l , r e r p e c t i v e l y . above. The Spec i f i c ac t i v i t y o r i g ina l l y and fo l lowing three recrystal l izat ion. ? n u C l I m l ,

REFERENCES

11. Fierer, L.M., and F ierer . M. (1967-1979) Reapents f o r Organic Synthesis. Yol. 1-7.

12. Spackmn. O.H.. Stein. Y . H . , and mare. 5. (1958) Anal. them. 30. 1190-1206 13. Langer, L.J., and tngel. L . L . (1958) J . 8101. C h m m - 5 8 8 14. Uerken, H . . and Haloway, C. (1956) J. B i o l . Chm. 223. 651-660 15. Lin-ver, H . . and Burk. 0. (1934) J . her. Chm. Sac. 56, 658-666 16. Lowry. O.H.. Rwebmugh, N.J., F a l l . A.L., and Randall. R.J. (1951) J. 8101. Chm.

17. Tmll, Y . . and Cannon, R.K. (1953) J . Bio l . Chm. 200, 803-811

John Yi ley 6 Sans, I n c . , Ne* York

193, 265-275

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Synthesis of 2-Bromoacetamidoestrone Methyl Ether and Study