Biochem. J. (1987) 245, 269-276 (Printed in Great Britain)

Affinity-labelling of the anti-inflammatory drug andprostaglandin-binding site of 3a-hydroxysteroid dehydrogenase ofrat liver cytosol with 17,8- and 21-bromoacetoxysteroidsTrevor M. PENNING, Kenneth E. CARLSON and Robert B. SHARPDepartment of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, U.S.A.

The homogeneous 3a-hydroxysteroid dehydrogenase of rat liver cytosol binds prostaglandins with lowmicromolar affinity at its active site and is competitively inhibited by the non-steroidal and steroidalanti-inflammatory drugs [Penning, Mukharji, Barrows & Talalay (1984) Biochem. J. 222, 601-611]. Toexamine the portion ofthis binding site that accommodates the glucocorticoid side chain, we have synthesized17,8-bromoacetoxy-5a-dihydrotestosterone (BrDHT) and 21-bromoacetoxydesoxycorticosterone (BrDOC)as affinity-labelling agents. Both these agents promote rapid inactivation of the purified enzyme in a time-and concentration-dependent manner. Analyses of the inactivation progress curves gave estimates of K1 forthe inactivators and half-life (t4) for the enzyme at saturation (r) as follows: Ki = 33 /SM and r = 18 s forBrDHT, and Ki = 10 /M and r = 203 s for BrDOC. Under initial-velocity conditions BrDHT and BrDOCact as competitive inhibitors, yielding Ki values identical with those measured in the inactivationexperiments. Both indomethacin and prostaglandin E2 can protect the enzyme from inactivation, yieldingKi values for these ligands consistent with those measured independently by competitive-inhibition studies.These data confirm that the bromoacetoxysteroids label the active site, which is coincident with theprostaglandin- and anti-inflammatory-drug-binding site. Neither gel filtration nor extensive dialysis restoresactivity to the enzyme inactivated with either affinity-labelling agent. Use of radioactive BrDHT or BrDOC,in which either the steroid portion is labelled with 3H or the bromoacetate portion is labelled with 14C,indicates that inactivation is accompanied by a stoichiometric incorporation of 0.7-1.0 molecules ofinhibitor per enzyme monomer. The linkage that forms between the dehydrogenase with either ["4C]BrDHTor [4C]BrDOC is stable to acid and base treatment. Complete acid hydrolysis of the enzyme inactivatedwith [14C]BrDHT, followed by amino acid analyses, indicates that 87% of the radioactivity is eluted withcarboxymethylcysteine. An almost identical result is obtained with [14C]BrDOC, where at least 75% of theradioactivity is eluted with this amino acid. Thus BrDHT and BrDOC alkylate at least one reactive cysteineresidue at the active site that may be of functional importance in binding the glucocorticoid side chain.

INTRODUCTION

The 3a-hydroxysteroid dehydrogenase (EC 1.1.1.50)of rat liver cytosol is an NAD(P)+-dependent oxido-reductase that catalyses the interconversion between3ac-hydroxy- and 3-oxo-steroids. Rat liver represents anabundant source of this protein, and milligram quantitiescan be readily purified (Penning & Talalay, 1983;Penning et al., 1984). A number ofdiverse functions havebeen assigned to the purified enzyme, and these include:reduction of 5a-dihydrotestosterone to 3az-androstane-diol (Hoff & Schreiffers, 1973), reduction of 5/,-dihydrocortisol to tetrahydrocortisol (Tomkins, 1956a,b)and, most recently, the oxidation of the isomeric pair oftrans-7,8-dihydrodiols derived from the metabolism ofbenzo[a]pyrene in vivo (Smithgall et al., 1986). Thus thepurified dehydrogenase catalyses the first step inandrogen metabolism, the second step in glucocorticoidmetabolism and oxidizes potent proximate carcinogens toas-yet-unidentified products.

A rather surprising feature of the purified 3ac-hydroxysteroid dehydrogenase is its potent inhibition byboth the non-steroidal and steroidal anti-inflammatorydrugs, which appear to bind at the active site (Penning& Talalay, 1983; Penning et al., 1984). Both these classesof drugs inhibit the enzyme in rank order of theirtherapeutic potency, and the IC50 (concentration causinghalf-maximal inhibition) values observed with many ofthese agents are lower than their peak plasma concen-trations. These earlier findings suggested that the enzymecould be used to predict the potency ofanti-inflammatorydrugs. Indeed, the ability to use the NADPH-linkedreduction of 5,-dihydrocortisone as a specific reaction toassay the enzyme in rat liver cytosol has led to thedevelopment of a rapid spectrophotometric screen fornon-steroidal-anti-inflammatory-drug potency (Penning,1985).The ability of the purified 3a-hydroxysteroid dehydro-

genase to be potently inhibited by anti-inflammatorydrugs has also led to the suggestion that the enzyme may

Vol. 245

Abbreviations used: 3a-hydroxysteroid dehydrogenase, 3a-hydroxysteroid:NAD(P)+ oxidoreductase (EC 1.1.1.50); androsterone, 5a-androstan-3a-ol-17-one; 5a-dihydrotestosterone, 5a-androstan-17,8-ol-3-one; BrDHT, 17fl-bromoacetoxy-5a-dihydrotestosterone (17fl-bromoacetoxy-Sa-androstan-3-one); desoxycorticosterone, 21-hydroxypregn-4-ene-3-one; BrDOC, 21-bromoacetoxydesoxycorticosterone (21-bromoacetoxy-pregn-4-en-3-one); prostaglandin E2, (5Z,1la,l3E,15S)-1 1,15-dihydroxy-9-oxoprosta-5,13-dien-1-oic acid; indomethacin, 1-(p-chlorobenzoyl)-5-methoxy-2-methylindol-3-ylacetic acid.

269

T. M. Penning, K. E. Carlson and R B. Sharp

be a target for these drugs in vivo (Pe g & Talalay,1983). The observation that the indomethacin-sensitivedehydrogenase is widely distributed in rat tissuessupports this hypothesis (Smithgall & Penning, 1985).The dehydrogenase is also competitively inhibited bylow-micromolar concentrations ofthe prostaglandins. Kivalues reported for the prostanoids range from 0.8 to12 ,M and are similar to Km values displayed by a varietyof prostaglandin dehydrogenases for their substrates(Nakano et al., 1969; Hansen, 1976; Yuan et al., 1980).We have some information that the dehydrogenasedisplays hydroxyprostaglandin dehydrogenase activity(T. M. Penning & R. B. Sharp, unpublished work).These findings may provide a link between the inhibitionof this enzyme by the anti-inflammatory drugs andregulation of prostanoid levels in inflammation.The abundance of the purified 3a-hydroxysteroid

dehydrogenase, its ability to predict the potency ofanti-inflammatory drugs and its ability to interconvertprostaglandins has prompted the commencement ofX-ray-crystallographic studies. In concert we have beenprompted to elucidate the topography of the enzymes'active site by using a classical affinity-labelling approach.The information generated from these studies mayprovide a better understanding of the architecture of ananti-inflammatory-drug- and prostaglandin-binding siteand may provide clues for the development of newanti-inflammatory agents.

It is recognized that the presence of the 17,8 side chainof glucocorticoids (17/.-21-dihydroxy-C-20-ketone) isessential for their anti-inflammatory activity (Wolff,1982). To examine the topography of the dehydrogenaseactive site that surrounds this side chain we havesynthesized two affinity-labelling agents that containalkylating groups in this portion of the steroid molecule,17,8-bromoacetoxy-5a-dihydrotestosterone (BrDHT)and 21-bromoacetoxydesoxycorticosterone (BrDOC).The selection of bromoacetoxysteroids was based on thetechnology developed by Warren and his co-workers(Sweet et al., 1972; Arias et al., 1973; Strickler et al.,1975), who have successfully affinity-labelled the activesite of the 3a,20,8-hydroxysteroid dehydrogenase ofStreptomyces hydrogenans with similar reagents.

In the present paper we evaluate BrDHT and BrDOCas affinity labels for the purified 3a-hydroxysteroiddehydrogenase. The amino acid(s) alkylated by theseagents at the prostaglandin- and anti-inflammatory-drug-binding site have been identified. This represents the firstreport of the affinity-labelling of a mammalian 3a-hydroxysteroid dehydrogenase.

MATERIALS AND METHODSAll steroids were purchased from Steraloids (Wilton,

NH, U.S.A.). [1,2,4,5,6,7(n)-3H]Dihydrotestosterone(135.0 Ci/mmol), [la,2a(n)-3H]desoxycorticosterone(48.0 Ci/mmol) and bromo[l-14C]acetic acid (56.0 Ci/mmol) were obtained from Amersham (ArlingtonHeights, IL, U.S.A.). [1-14C]Glycollic acid (calciumsalt; 40.0 mCi/mmol) was a product of ICN Radio-chemicals (Irvine, CA, U.S.A.). Prostaglandin E2 was aproduct of Bio-Mol, Philadelphia, PA, U.S.A. Indo-methacin was purchased from Sigma Chemical Co. (St.Louis, MO, U.S.A.), and Sephadex G-25F was a productofPharmacia (Piscataway, NJ, U.S.A.). Pyridine ('Gold-label'), [2H]chloroform, tetramethylsilane and dicyclo-

hexylcarbodi-imide were purchased from AldrichChemical Co. (Milwaukee, WI, U.S.A.). All solvents wereof lLV. grade and were obtained from Burdick-Jackson(Media, PA, U.S.A.). Budget-Solve scintillation fluid waspurchased from Research Products International (MountPleasant, IL, U.S.A.).

All n.m.r. spectra were recorded on a Brucker WM-360MHz spectrometer, in [2Hlchloroform with a deuteriumfield lock relative to tetramethylsilane. I.r. spectra wererecorded in chloroform on a Perkin-Elmer 521 spectro-meter with an extended range interchange. Absorptionbands are listed as strong (s), medium (m), weak (w)and broad (b). Reactions were monitored by t.l.c.conducted on silica-gel GF 250,m plates(10 cm x 2.5 cm) (Analtech, Newark, DE, U.S.A.).

Steroid synthesisSynthesis of 17p-bromoacetoxy-5a-dihydrotestosterone.

Sa-Dihydrotestosterone (0.2 mmol) was dissolved in3.0 ml of methylene chloride (CaH-distilled). Themixture was stirred on ice and 0.2 mmol of bromoaceticacid dissolved in 1.0 ml of methylene chloride was thenadded. Upon further stirring a slight molar excess ofdicyclohexylcarbodi-imide in 1.0 ml of methylene chlor-ide was added to initiate the reaction. After 5 min on icethe mixture turned cloudy, indicating the precipitation ofsome dicyclohexylurea; at this time a catalytic quantity ofpyridine (10 1) was added and the mixture was stirred onice for a further 120 min. The reaction was monitored byt.l.c. in benzene/ethyl acetate (4:1, v/v), and the plateswere sprayed with methanolic H2SO4. Under theseconditions the starting material had an RF of 0.27,whereas the product had an RF of 0.62. Once thereaction was complete, the solvent was removed under astream of N2 and the residue was dissolved in 4.0 ml ofcold acetone. The insoluble dicyclohexylurea wasremoved by filtration and the filtrate which contained theproduct was evaporated to dryness in vacuo. Theproduct was recrystallized from the residue in methanol.I.r.: (cm-'), absence of 0-H stretch at 3600 cm-' (b);C=O stretch at 1690 (s) with shoulder at 1650;C-C(=O)-O stretch at 1275 (s) and O-C-C stretch at1100 (s). N.m.r. :6(p.p.m.)([2H]chloroform)0.80(3 s,C-18-CH3), 1.05 (3 s, C-19 -CH,3), 3.58 (2 s, Br-CH2-C=O)and 4.70 (lt, C-17 -CH).

Synthesis of 21-bromoacetoxydesoxycorticosterone.This compound was synthesized in a similar manner tothat described for the synthesis of 17,-bromoacetoxy-Sa-dihydrotestosterone. The reaction was monitored byt.l.c. as described above; the starting material had anRF of 0.16, and the product had an RF of 0.43.Recrystallization from methanol afforded yellow floccu-lent crystals. I.r.: (cm-') absence of 0-H stretch at3500 cm-' b, and O-C-C stretch at 1100 (s). Theadditional CO stretch of the ester was hidden withinthe existing carbonyl stretches. N.m.r.: a (p.p.m.) ([2H]chloroform), 0.70 (3 s, C-18 -CH3), 1.06 (3 s, C-19 -CH3).4.0 (2 s, Br-CH2-C0=), 4.6 (ld, C-21 -CH2), 4.8 (ld,C-21 -CH2) and 5.75 (1 s, C-4 -CH). U.v. Amax. 240 nm(e= 17000 M.cm-').

Synthesis of 13H117.8-bromoacetoxy-Sa-dihydrotesto-sterone. This synthesis was conducted in the mannerdescribed above by using 25 mg of [3H]5a-dihydrotesto-sterone diluted to a specific radioactivity of 0.63 mCi/

1987

270

Affinity-labelling of 3a-hydroxysteroid dehydrogenase

mmol. The specific radioactivity ofbromoacetoxy-5Sa-dihydrotestosteronerecrystallizations was 495 c.p.m./nmol.

the r3H]17f-after three

Synthesis of 114Cj17I-bromoacetoxy-5a-dihydrotesto-sterone. This synthesis was conducted in the mannerdescribed above by using 14.5 mg of unlabelled steroidand 6.95 mg of bromo[I-14Clacetate diluted to a specificradioactivity of 5.0 mCi/mmol. The specific radioactivityof the [14C] I 7f-bromoacetoxy-5a-dihydrotestosteroneafter three recrystallizations was 6560 c.p.m./nmol.

Synthesis of [3Hj21-bromoacetoxydesoxycortico-sterone. This synthesis was conducted in the mannerdescribed above by using 28.4 mg of labelled steroid,[3H]desoxycorticosterone diluted to a specific radio-activity of 2.3 mCi/mmol. The specific radioactivity of[3H]21-bromoacetoxydesoxycorticosterone after threerecrystallizations was 2313 c.p.m./nmol.

Synthesis of 1P4C]21-bromoacetoxydesoxycortico-sterone. This synthesis was conducted in themanner described above by using 16.5 mg of unlabelledsteroid and 6.95 mg of bromo[I-14C]acetate dilutedto a final specific radioactivity of 5.0 mCi/mmol. Thespecific radioactivity of 21-bromo[1-14C]acetoxydesoxy-corticosterone after three recrystallizations was6766 c.p.m./nmol.

Enzyme studiesPurification of 3a-hydroxysteroid dehydrogenase. 3a-

Hydroxysteroid dehydrogenase was purified to homo-geneity from male Sprague-Dawley-rat liver cytosol asdescribed by Penning et al. (1983). The final specificactivity was 1.5,mol of androsterone oxidized/min permg under standard assay conditions (see below).

Standard enzyme assay. The standard spectrophoto-metric assay for the 3a-hydroxysteroid dehydrogenasewas conducted in 1.0 ml systems containing: 75 sM-

androsterone, 2.3 mM-NAD+, 4% (v/v) acetonitrile and100 mM-potassium phosphate buffer, pH 7.0. Thereaction was initiated by the addition of the enzyme andthe absorbance of the reduced nicotinamide (NADH)nucleotide was measured at 340 nm on a Gilford260 UV/Vis recording spectrophotometer at 25 'C. Theabsorbance change was monitored over 5 min andconverted into nmol of product formed/min by using ane value of 6270 M-1 cm-' for NADH. Under theseconditions non-enzymic rates were negligible.

Inactivation experiments. Purified 3a-hydroxysteroiddehydrogenase (0.169 mg) was diluted to 1.0 ml anddialysed overnight against three changes of 10 mm-potassium phosphate, pH 7.0, containing 1 mM-EDTA.The enzyme was divided into 100 1l portions and theinactivation event was initiated by the addition ofbromoacetoxysteroid to give final concentrations ofinhibitor that ranged from 2.5 to 50/M in 6%acetonitrile. The enzyme solution was then incubated at25 'C, and at various times 2-10l,l samples wereremoved and diluted into the standard enzyme assay of1.0 ml. Because of these substantial dilutions (100-500-fold), any further alkylation events are effectivelyquenched. The determination of enzyme activity in theseassays was thus a measure of enzyme inactivation. Plots

Vol 245

of percentage activity remaining versus time gaveestimates of the half-life of the enzyme (t1). These datawere further analysed to give estimates of the ti atsaturation (Xr) and Ki (dissociation constant for thereversible enzyme-inhibitor complex). For further detailsof the analyses, see the Results section.

Protection studies. In these experiments a fixedconcentration of BrDHT or BrDOC was used toinactivate the homogeneous 3a-hydroxysteroid de-hydrogenase. Incubations were then repeated in thepresence of increasing concentrations of protectingligand (e.g. indomethacin or prostaglandin E2). From thevarious progress curves, estimates of the initial velocityof inactivation were made and plotted against theconcentration of protecting ligand in a manner formallyanalogous to the Dixon plots used for the analyses ofcompetitive-inhibition data. From the slope of theseplots the Ki values for the protecting ligand wascomputed (Penning et al., 1981).

Competitive-inhibition studies. Ki values for com-petitive inhibitors were derived from initial-velocitymeasurements in which the concentration of inhibitorwas varied over three fixed substrate (androsterone)concentrations. Primary Dixon plots were constructedand the slopes of these lines were then plotted against thereciprocal of the substrate concentration to yield asecondary plot. The Ki for the competitive inhibitor wascalculated from the slope of the secondary plot asdescribed by Siegel (1975).

Amino acid analyses. Large quantities of enzyme(10.0-15.0 nmol, M, 34000) were inactivated withbromo[14C]acetoxysteroid. Excess radiolabelled steroidwas removed by gel-exclusion chromatography, and thepeak protein fractions were pooled, checked forstoichiometry by liquid-scintillation counting and dia-lysed against three changes of distilled water overnight.The aqueous samples were then freeze-dried and thedried residues were hydrolysed in 6 M-HCI for 24 h. Theacid was removed by drying in vacuo and the residue wastaken up in sodium citrate buffer, pH 2.2, for amino acidanalysis on a Beckman 6300 high-performance aminoacid analyser. After amino acid detection with ninhydrin,the effluent was collected at 12 s intervals. Fractions werethen dissolved in Budget-Solve and radioactivityquantified by liquid-scintillation counting. Calibration ofthe dead volume between the detector and fractioncollector was achieved by using a cysteic acid standard.It was estimated that the time interval that existsbetween the detector and fraction collector was approx.12 s, or one fraction. This knowledge permitted accuratesuperimposition of the radioactive scan over theninhydrin trace.

Radioactivity measurements. 3H and 14C radioactivitywere counted on a Tracor 43 analytic liquid-scintillationcounter, which has a machine efficiency of 53% for 3Hand 95% for 14C. All samples were measured ascorrected c.p.m. Aqueous samples were counted forradioactivity in Budget-Solve, and organic samples werecounted for radioactivity in a toluene-based scintillation

271

T. M. Penning, K. E. Carlson and R. B. Sharp

fluid [4 g of 2,5-diphenyloxazole (PPO) and 200 mgof 1,4-bis-(4-methyl-5-phenyloxazol-2-yl)benzene (di-methyl-POPOP/litre of toluene].

RESULTSInactivation of homogeneous 3a-hydroxysteroiddehydrogenase by bromoacetoxysteroids

Previous studies indicated that the homogeneous3a-hydroxysteroid dehydrogenase is sensitive to com-petitive inhibition by steroidal and non-steroidal anti-inflammatory drugs (Penning & Talalay, 1983; Penninget al., 1984). Knowing that a 17, side chain (17/,-21-dihydroxy-C-20-ketone) is required for glucocorticoidactivity (Wolff, 1982), steroids containing a bromoacet-oxy moiety either at C-17 or C-21 were synthesized (e.g.BrDHT and BrDOC) as affinity-labelling agents. Thesecompounds have the potential to label that part of thedehydrogenase active site concerned with the accom-modation of the glucocorticoid side chain. Both BrDHT

r-

0

(U

.tco

ENc._

and BrDOC inactivate the purified 3a-hydroxysteroiddehydrogenase (Figs. la and lb) in a rapid time-dependent manner. Semilogarithmic plots of enzymeactivity remaining versus time show that ti values can beas short as a few minutes. At lower concentrations ofinactivator, the progress curves for inactivation showmarked curvature, attributable to the almost stoichio-metric amounts of enzyme and inhibitor present in theincubation. At higher concentrations of inactivator theprogress curves are almost linear and appear to indicatepseudo-first-order kinetics. In analysing this data it isassumed that, in each progress curve, the initial velocityof enzyme inactivation is a function of k[EI], where k isa pseudo-first-order rate constant and [El] is theconcentration of the enzyme-inactivator complex. Toobtain estimates of these pseudo-first-order rate con-stants (ka . values), tangents were drawn to the progresscurves. TMs method, if anything, yields underestimatesof the true velocity of inactivation. Analyses of adouble-reciprocal plot of 1 /kapp. versus 1/[I] gave ameasure of 1 /k+2 (limiting rate constant for inactivation)

Time (min)

0 0.1 0.2 -0.1 0 0.1 0.21/[1] (#M-,)

Fig. 1. Inactivation of homogeneous 3a-hydroxysteroid dehydrogenase with BrDHT and BrDOC

Homogeneous 3a-hydroxysteroid dehydrogenase (0.49 nmol) in 100,ll of 10 mm-potassium phosphate, pH 7.0, containing1 mM-EDTA was incubated at 25 °C in the presence of increasing concentrations of either BrDHT (2.5-25 ,SM) (a) or BrDOC(2.5-30 /uM) (b). Bromoacetoxysteroids were dissolved in 6% acetonitrile. Over the time course indicated, 2-10 ,1 samples were

removed from the incubation medium and diluted directly into the standard enzyme assay, and the amount of enzyme activityremaining was determined (see the Materials and methods section). Rate constants for inactivation were calculated by drawingtangents to the inactivation progress curves. These estimates of the pseudo-first-order rate constants were plotted against theconcentration of the inactivator to yield the double-reciprocal plot for the inactivation of 3a-hydroxysteroid dehydrogenaseby either BrDHT (c) or BrDOC (d).

1987

250

,,, 200 -

l 150 -

- 100 /

50 (c)I I

-0.1

272

Affinity-labelling of 3a-hydroxysteroid dehydrogenase

and 1/Ki (dissociation constant for the El complex).Generation of the straight lines shown in Figs. 1(c) and1(d) indicates that, with both BrDHT and BrDOC, theprocess of inactivation is saturable and thereforedependent on the total concentration of enzyme. Datatransformed in this manner yields the following kineticconstants: Ki = 33.0 /M and k+2= 40.0 x l0-3 s- forBrDHT, and Ki = 10.0 /M and k+2 = 3.0 x 10-3 s-' forBrDOC. These limiting rate constants predict that, atsaturation, exceedingly short half-lives exist for theenzyme. Thus the tq for the enzyme when saturated withBrDHT is 18 s and that with BrDOC is 203 s.

It should be emphasized that it is not possible tochange the conditions of the inactivation experimentsuch as to reduce the curvature observed in the progresscurves. This would require either decreasing theconcentration of the enzyme or increasing the concen-tration of the steroid inactivator in the incubation;neither is possible. At lower concentrations of enzyme itwould not be possible to measure enzyme activityaccurately at the end of the experiment. At higherconcentrations of steroid the inactivators are insoluble.

Evidence that BrDHT and BrDOC label theprostaglandin- and anti-inflammatory-drug-binding siteof 3a-hydroxysteroid dehydrogenaseUnder initial-velocity conditions in which the concen-

tration of androsterone (75 pM) and nicotinamidenucleotide (2.3 mM) are saturating, the bromoacetoxy-steroids can act as competitive inhibitors of catalyticquantities of enzyme (Figs. 2a and 2b). Under suchconditions the bromoacetoxysteroids presumably bindto the enzyme in a rapid reversible fashion, but areprevented from initiating an alkylating event bysubstrate. In these competitive-inhibition studies the K1values determined for the bromoacetoxysteroids aresimilar to those determined in the inactivation experi-ments. These kinetic analyses suggest that the bromo-acetoxysteroids label the active site.

Micromolar concentrations of both indomethacinand prostaglandin E2 also protect the enzyme frominactivation mediated by high concentrations of thebromoacetoxysteroids [Figs. 3(a) and 3(b) (only the dataobtained with BrDHT as inactivator are shown)].Although complete protection with either indomethacinor PGE2 is only observed at concentrations greater than100 /M, it should be emphasized that, in these experi-ments, this represents only a 10-20 fold excess over theconcentration of enzyme and a meagre 2-3-fold excessover the concentration of inactivator. These experimentssuggest that powerful protection with these ligands isbeing achieved. Ki values obtained for indomethacin andPGE2 from these protection studies are comparable withthose measured directly for these ligands in competitive-inhibition studies (Table 1). These kinetic data indicatethat the bromoacetoxysteroids affinity-label the anti-inflammatory-drug- and prostaglandin-binding site ofthe enzyme, which appears to be coincident with theactive site.

Evidence that inactivation of the 3a-hydroxysteroiddehydrogenase results in covalent attachment of thesteroid

Several lines of evidence suggest that BrDHT andBrDOC covalently modify the dehydrogenase active site.In experiments in which the dehydrogenase is inactivated

Vol. 245

with either BrDHT or BrDOC to within less than 5% ofits initial activity, enzyme activity cannot be restoredafter substantial (100-fold) dilution into the assaysystem. Further, enzyme activity is not restored afterextensive dialysis against 10 mM-potassium phosphate,pH 7.0, containing 1 mM-EDTA or by gel filtration.During these manipulations control enzyme retains fullactivity.

Stoichiometric incorporation of radiolabelledbromoacetoxysteroids into the active site of3a-hydroxysteroid dehydrogenase

Radioactive probes were synthesized in which eitherthe steroid nucleus of BrDHT and BrDOC was labelledwith 3H or the bromoacetoxy portion of BrDHT andBrDOC was labelled with 14C. When these agents were

-12 -8 -4 0 4 8 12 16 20[Inhibitor](#M)

Fig. 2. Competitive inhibition of 3a-hydroxysteroid dehydro-genase by BrDHT and BrDOC

Initial velocities of androsterone oxidation were measuredat three fixed substrate concentrations (15-75.0 ,sM) whilethe concentrations of either BrDHT (0-40,uM) (a) orBrDOC (0-20 uM) (b) were varied. In each instance theassays were performed under the standard conditionsdescribed in the Materials and methods section. The finalconcentration of organic solvent was 6% acetonitrile.

273

T. M. Penning, K. E. Carlson and R. B. Sharp

0

Time (min)'

5 10 15 20 25 30

-40 -20 0 20 40 60

[ Indomethacin] (#M)

80 100

800-

600-

.1,400

(d)

-120 -80 -40 0 40 80 120

[Prostaglandin E21 (SAM)

Fig. 3. Indomethacin and prostaglandin E2 protect 3a-hydroxysteroid dehydrogenase from inactivation mediated by BrDHT

Homogeneous 3a-hydroxysteroid dehydrogenase (0.32 nmol) in 100 pl of 10 mM-potassium phosphate, pH 7.0, containing1 mM-EDTA, was incubated with 10.0 1sM-BrDHT in 6% acetonitrile in the presence of increasing amounts of indomethacin(0-100 ,uM). At various times, 2-10,ul samples were removed and diluted into the standard enzyme assay mixture. The amountof enzyme activity remaining was determined (a) after correcting the initial velocities for the inhibition due to the presence ofindomethacin in the enzyme assay. These corrections were made from the appropriate dose-response curve. In a similar setof experiments, homogeneous enzyme (1.0 nmol) in 100 #1 was incubated in the presence of 50 ,#M-BrDHT in the presence ofincreasing amounts of prostaglandin E2 (0-144 /LM). At various times, 2-10 #1 samples were removed and diluted into thestandard enzyme assay mixture, and the amount of enzyme activity remaining was determined (b). The final concentration ofprostaglandin E2 present in these assays had no effect on the initial velocities observed. Rate constants for inactivation were

calculated by drawing tangents to the inactivation progress curves. These estimates of the pseudo-first-order rate constants were

plotted against the concentration of indomethacin (c) or prostaglandin E2 (d); slopes from these secondary plots were used tocalculate the Ki values for indomethacin and prostaglandin E2.

used to inactivate the dehydrogenase, subsequentgel-exclusion chromatography to remove excess steroidindicated that the inactivation event was accompanied bythe incorporation of 0.7-1.0 molecules of inactivator perenzyme monomer (results not shown). This suggests thata discrete amino acid is being labelled at the active siteby both the steroid and bromoacetoxy moieties ofBrDHT and BrDOC. Since the same stoichiometry isobserved with all four probes, this suggests that tr-ansferof the carboxymethyl group with subsequent release ofthe steroid is not facilitated by nucleophilic amino acidsat the active site.

Stability of the covalent linkage that forms between the_xysactroxystroids (BrDHT and BrDOC) and3.-hydroxysteroid dehydrogenase

In these experiments 6.5-12.0 nmol of homogeneous3ix-hydroxysteroid dehydrogenase were inactivated with

either [14C]BrDHT or [14C]BrDOC; excess steroidwas removed by gel filtration, and the[t4C]carboxymethylated enzyme was subjected to avariety of treatments to establish the stability of thecovalent linkage. With either preparation, the inactivatedenzyme retained its label when exposed to extremes ofacidity. The covalent bond was somewhat less stable inalkali. This demonstrates that the carboxy group ofaspartate or glutamate was not carboxymethylated, sincethe resulting ester should have been cleaved under theacid conditions employed with subsequent release ofradiolabel.

Evidence that covalent modification of the3a-hydroxysteroid dehydrogenase results in a largeconformational changeThe u.v. spectrum of the homogeneous dehydrogenase

is not unusual and shows a Amax at 280 nm attributable

1987

4-

0

0s

E

C

w

100

80

60

40

20

10

0

'- I I I I

1000

'T800 -

- 600 -

400

(c)

274

Affinity-labelling of 3a-hydToxysteroid dehydrogenase

Table 1. Comparison of dissociation constants (Kd values)determined for indomethacin and prostaglandin E2 byvarious methods

with cysteic acid or glycollic acid, and it is also unlikelyto be thioglycollic acid, since the unidentified peak has ashorter retention time than that compound..

Dissociation constants for the competitive inhibitorsindomethacin and prostaglandin E2 were determined fromDixon plots using androsterone as substrate (as describedin the Materials and methods section). These constants werealso derived from inactivation experiments in whichvarious concentrations of either indomethacin (0-100 /LM)or prostaglandin E2 (0- 150sM) were used to protecthomogeneous 3a-hydroxysteroid dehydrogenase frominactivation by either 50 ,tM-BrDOC or 10 /sM-BrDHT.

Competitive KdMethod of derivation inhibitor (UM)

Dixon plot Indomethacin 1-3Prostaglandin E2 10.0

Protection studies Indomethacin 6.0with BrDHT Prostaglandin E2 5.0

Protection studies Indomethacin 6.0with BrDOC Prostaglandin E2 7.0

to the absorbance of the aromatic amino acids as well asa large end absorbance due to, the presence of the peptidebonds. After inactivation with BrDHT the absorbance ofthe aromatic amino acids appears to be unaffected;however, the end absorbance undergoes a substantial redshift accompanied by a 4-5-fold increase in intensity.Similar changes are observed in the u.v. after inactivationof the purified dehydrogenase with BrDOC. Since theabsorbance of the aromatic amino acids is unperturbedby inactivation with either BrDHT or BrDOC, theseamino acids would appear to be distant from thesteroidal ligands. The large change in absorbanceobserved in the far u.v. may suggest that a substantialconformational change occurs as a result of theinactivation event.

Identification of the amino acid alkylated by BrDHTand BrDOC

Inactivation of a large quantity of homogeneousdehydrogenase with [14C]BrDHT followed by subsequentremoval of excess steroid by gel-exclusion chromato-graphy afforded a sample that was freeze-dried andsubjected to complete acid hydrolysis in 6 m-HCl for 24 h.The sample was then subjected to amino acid analysesand the effluent from the analyser was collected forliquid-scintillation counting. It was found that most ofthe radioactivity (85-87% of the total) was associatedwith a single amino acid with a retention time of 6.0 min(Fig. 4). This amino acid is co-eluted with carboxy-methylcysteine. An almost identical result wasobserved from four separate analyses of [14C]BrDHT-inactivated dehydrogenase.When enzyme inactivated with [14CIBrDOC was

subjected to a similar procedure, most of the radioactivity(75-77%) was also found to be co-eluted with carboxy-methylcysteine (results not shown). This result suggeststhat both affinity ligands predominantly label cysteineresidue(s) at the active site.With both labels the remainder of the radioactivity

(13-25%) appears to be associated with a ninhydrin-negative peak that is eluted at 2.4 min. At the presenttime this peak remains to be identified; it is not co-eluted

A

A

0 la 2D 30 4ETinm- (min)

Fig. 4. Identification of the amino acid alkjted byF4CJBrDHT

Homogeneous 3a-hydroxysteroid dehydrogenase(21.0 nmol/1.5 ml) wasinactivated by 9&% by 100 nmol of['4C]BrDHT as descnrbed in the text. Excess steroid wasremoved by gel-exclusion chromatography, the radio-labelled protein fractions, which contained one mlculeof steroid/enzyme monomer, were pooled and: dialysedagainst water. An aqueous sample was prepared andsubjected to amino acid analysis as described in theMaterials and methods section. (a) Ninhydrin trace ( )and 14C radioactivity (--)of a portion aof thehydrolysed sample; (b) "'C radioactivity detected afteramino acid analysis of a second portion of the hydrlysedsample; (c) ninhydrin trace of 10.0 nmol of cysteic acidand carboxymethylcysteine standards ( ) plus radio-active detection of a [14CJglycollic acid standard (

Vol. 245

2a's

276 T. M. Penning, K. E. Carlson and R. B. Sharp

DISCUSSIONThe present paper provides the first successful account

of the affinity labelling of the active site of a mammalian3a-hydroxysteroid dehydrogenase. This enzyme is ofinterest, since it is involved in both androgen (Hoff &Schreiffers, 1973) and polycyclic-aromatic-hydrocarbon(Smithgall et al., 1986) metabolism. One novel propertyof the enzyme is its potent inhibition by prostaglandinsand anti-inflammatory drugs (Penning & Talalay, 1983).The abundant supply of this enzyme in rat liver cytosolindicates that it represents a useful model for mappingthe topography of an anti-inflammatory-drug- andprostaglandin-binding site.We have employed BrDHT and BrDOC to label

amino acid residues that may be of functional importancein accommodating the glucocorticoid side chain at theactive site. The kinetic data presented indicate that bothagents label the appropriate site. The rates of inactiva-tion observed are exceptionally high; other studies inwhich bromoacetoxysteroids were used to affinity-labelhydroxysteroid dehydrogenases indicate that half-livesgenerally range from 20 min to 10 h (Sweet et al., 1972;Arias et al., 1973; Strickler et al., 1975). Radiolabelledprobes indicate that a stoichiometric incorporation oflabel accompanies the inactivation of 3a-hydroxysteroiddehydrogenase. This is observed irrespective of whetherthe steroid nucleus or the bromoacetate group is radio-labelled. With each probe carboxymethylcysteine is theamino acid predominantly labelled. Whether bothBrDHT and BrDOC label an identical cysteine residueremains to be determined. This will require proteolyticdigestion and peptide mapping of enzyme inactivatedwith either BrDHT or BrDOC.The use of bromoacetoxysteroids to label the active

site of hydroxysteroid dehydrogenases was originallydescribed by Warren and his colleagues (Sweet et al.,1972; Arias et al., 1973; Strickler et al., 1975). In thoseearlier studies the 3a/20,-hydroxysteroid dehydrogenaseof Streptomyces hydrogenans was affinity-labelled with6fl, 1 la- or 16a-bromoacetoxyprogesterone, and thecarboxymethylated amino acids identified were shown tobe cysteine, methionine and histidine respectively. Withthe use of a 16a-bromoacetoxyprogesterone, the activesite of the 17fl/20a-hydroxysteroid dehydrogenase ofhuman placenta has also been affinity-labelled (Strickleret al., 1981). In this particular instance a dicarboxy-methylated histidine was the result of the alkylationevent. On the basis of studies with 16-bromoacetoxy-progesterone, a histidine residue would appear to beimportant in binding the steroid D-ring at the active siteof 20a- and 20,8-hydroxysteroid dehydrogenase. Thispresent study demonstrates that a cysteine residue maybe important in binding the steroid side chain at the3a-hydroxysteroid dehydrogenase active site.

Although we have used a classical approach in ourstudies, this is the first time such agents have been usedto probe the structure of a prostaglandin- and/oranti-inflammatory-drug-binding site. The linkage thatforms between either BrDHT or BrDOC appears to beexceptionally stable to acid and somewhat stable toalkali. Such stability suggests that both probes could beused with success to isolate and sequence an active-sitepeptide of the dehydrogenase. This information willprovide further clues to the architecture of a binding sitecapable of binding proximate carcinogens (trans-dihydrodiols of polycyclic aromatic hydrocarbons),prostaglandins and anti-inflammatory drugs.

This work was supported by National Institutes of HealthGrant GM 33464. K.E.C. was supported by Training GrantGM 07229. Amino acid analyses were conducted at theUniversity of Pennsylvania Protein Chemistry Laboratory,which is supported in part by a University BRSG grant,RR-05415, a Medical School BRSG grant, R-07083, and aResearch Resources Equipment Grant, RR-01412. We thankMs. Caryn Glusman for expert technical assistance in runningthe amino acid analyser.

REFERENCESArias, F., Sweet, F. & Warren, J. C. (1973) J. Biol. Chem. 248,

5641-5647Hansen, H. S. (1976) Prostaglandins 12, 647-679Hoff, H. G. & Schreiffers, H. (1973) Hoppe-Seyler's Z. Physiol.

Chem. 354, 507-513Nakano, J., Anggard, E. & Samuelsson, B. (1969) Eur. J.

Biochem. 11, 386-389Penning, T. M. (1985) J. Pharm. Sci. 74, 651-654Penning, T. M. & Talalay, P. (1983) Proc. Natl. Acad. Sci.

U.S.A. 80, 4505-4508Penning, T. M., Covey, D. F. & Talalay, P. (1981) Biochem. J.

193, 217-227Penning, T. M., Mukharji, I., Barrows, S. & Talalay, P. (1984)

Biochem. J. 222, 601-611Siegel, I. H. (1975) Enzyme Kinetics, pp. 100-125, Wiley-

Interscience, New YorkSmithgall, T. E. & Penning, T. M. (1985) Biochem. Pharmacol.

34, 831-835Smithgall, T. E., Harvey, R. G. & Penning, T. M. (1986)

J. Biol. Chem. 261, 6184-6191Strickler, R. C., Sweet, F. & Warren, J. C. (1975) J. Biol. Chem.

250, 7656-7662Strickler, R. C., Tobias, B. & Covey, D. F. (1981) J. Biol.Chem. 256, 316-321

Sweet, F., Arias, F. & Warren, J. C. (1972) J. Biol. Chem. 247,3424-3428

Tomkins, G. (1956a) J. Biol. Chem. 218, 437-447Tomkins, G. (1956b) Recent Prog. Horm. Res. 12, 125-183Wolff, M. E. (1982) in Burger's Medicinal Chemistry (Wolff,M. E., ed.), 4th edn., vol. 3, pp. 1273-1316, Wiley, New York

Yuan, B., Tai, C. L. & Tai, H.-H. (1980) J. Biol. Chem. 255,7439-7443

Received 29 October 1986; accepted 23 March 1987

1987