CHIRALITY 3:104-111 (1991)

Benzylic Alcohols as Stereospecific Substrates and Inhibitors for Aryl Sulfotransferase SATISH I. RAO AND MICHAEL W. DUFFEL Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa, Iowa City, Iowa 52242

ABSTRACT Aryl sulfotransferase IV catalyzes the 3’-phosphoadenosine-5’phosphosulfate (PAPSI-dependent formation of sulfuric acid esters of benzylic alcohols. Since the benzylic carbon bearing the hydroxyl group can be asymmetric, the possibility of stereochemical control of substrate specificity of the sulfotransferase was investigated with benzylic alcohols. Benzylic alcohols of known stereochemistry were examined as potential substrates and inhibitors for the homogeneous enzyme purified from rat liver. For 1-phenylethanol, both the ( + )-(R)and ( - )-(S)-enantiomers were substrates for the enzyme, and the kc,,/&, value for the ( - 1-(S)-enantiomer was twice that of the ( + )-(Rbenantiomer. The enzyme displayed an absolute stereospecificity with ephedrine and pseudoephedrine, and with 2-methyl-1-phenyl-1-propanol;that is, only ( - )-( lR,ZS)-ephedrine, ( - )-(lR,2R)-pseudoephedrine, and ( - )-(S)-2-methyl-l-phenyl-l-propanol were substrates for the sulfotransferase. In the case of 1,2,3,4-tetrahydro-l-naphthol, only t h e (-)-(R)-enantiomer was a substrate for t h e enzyme. Both ( + )-(R)-2-methyl-l-phenyl-l-propanol and ( + )-(S)-1,2,3,4-tetrahydro-l-naphthol were competitive inhibitors of the aryl sulfotransferase-catalyzedsulfation of 1naphthalenemethanol. Thus, the configuration of the benzylic carbon bearing the hydroxyl group determined whether these benzylic alcohols were substrates or inhibitors of the rat hepatic aryl sulfotransferase IV. Furthermore, benzylic alcorepresent a new class of inhibhols such as ( + )-(S)-l,2,3,4-tetrahydro-l-naphthol itors for the aryl sulfotransferase.

KEY WORDS: stereochemistry, sulfation, enantioselectivity, inhibition, chiral, mechanism, stereoselective INTRODUCTION

Stereoselective metabolism of drugs, carcinogens, and other xenobiotics is gaining increased recognition as an important factor in both the detoxication and metabolic activation of these substances in biological systems. Several aspects of stereoselective drug metabolism have been recently reviewed,’-5 and studies on the role of stereochemistry in the specificity of drug metabolizing enzymes have been carried out with varied breadth for cytochromes P-450,5-10 microsomal FAD-containing m~nooxygenase,’~-~~ epoxide hydroUDP-glucuronyltransferases,1s-20 and glutathione t r a n ~ f e r a s e s . ~ ’Although -~~ there have been studies on stereochemical considerations in the sulfation of phenolic oxygen atoms within chiral molecule^,^^-^^ there have been no previous investigations of structure-activity relationships for the sulfation of chiral benzylic alcohols. Many benzylic alcohols are encountered as direct metabolic products obtained by cytochrome P-450 monooxygenase-catalyzed stereoselective hydroxylation of xenobiotics. In some cases this benzylic hydrox0 1991 Wiley-Liss, Inc.

ylation, and subsequent formation of chemically reactive sulfuric acid esters, leads to ultimate carcinogens capable of covalent binding to nucleic acids and proteins; representative examples include safr01e,~l 2’,3’-dehydroe~tragole,~~ 7,12-dimethylbenz[aland 5 a n t h r a ~ e n e ,6-methylben~o[a]pyrene,~~ ~~ rnethylchry~ene.’~ For some benzylic alcohols such as 2-methylbenzylalcoho1,36benzyl and 1naphthalenemethan01,3~sulfation is a detoxication reaction that leads either to a subsequent reaction with glutathione (resulting in the formation of glutathione conjugates and mercapturic acids), or to excretion of the unchanged sulfuric acid ester. Numerous drugs are also metabolized via benzylic oxidation, although the importance of sulfation in the toxicity or detoxication of the resulting benzylic alcohols has not yet received extensive investigation. Representative examples of drugs undergoing hydroxylation at a benzylic carbon Received for publication October 2, 1990; accepted November 15, 1990. Address reprint requests to Michael W. Duffel at the address given above.

STEREOSPECIFICITYOF ARYL SULFOTRANSFERASE IV

include t ~ l b u t a m i d e , ,metoprol01,~~ ~ amitriptyline;' and n ~ r t r i p t y l i n e . ~ ~ A fundamental step in determining the role of sulfation in the metabolism of benzylic alcohols, and in the biotransformation of those xenobiotics metabolically converted to benzylic alcohols, is a n understanding of the substrate specificity and stereoselectivity of sulfotransferases that catalyze these reactions. One sulfotransferase that catalyzes formation of sulfuric acid esters of benzylic alcohols is aryl sulfotransferase IV.42,43Aryl sulfotransferase IV (EC 2.8.2.9) is one of at least four isoenzymes of phenol sulfotransferase purified from male rat l i ~ e r . Isoenzyme ~ - ~ ~ IV catalyzes the formation of sulfuric acid esters from a wide variety of phenols, hydroxamic acids, catecholamines, tyrosine carboxylesters, peptides with N-terminal tyrosines, and benzylic a l ~ o h o l s . Studies ~ ~ , ~ ~ on the substrate specificity of the enzyme for benzylic alcohols have indicated that apparent K, values decrease with increasing lipophilicity of the ~ u b s t r a t e . ~Since , there was no the correlation effect of substrate lipophilicity on V,, between apparent K , and lipophilicity of the benzylic alcohol was interpreted as indicative of lipophilic interactions in binding of the benzylic alcohol to the enzyme.43In addition to these effects of lipophilicity on the apparent K,, the catalytic efficiency (kcat/Km)of the aryl sulfotransferase with the ( - )-(S)-enantiomer of 1- phe nyl e t ha no l was h i g h e r t h a n with t h e ( + ) - ( R ) - e n a n t i ~ m e rThis . ~ ~ was the first indication of stereoselectivity in the sulfation of benzylic alcohols catalyzed by aryl sulfotransferase IV. The results presented herein describe the first structure-activity studies on the stereochemistry of benzylic alcohol sulfation catalyzed by a purified aryl sulfotransferase.

105

Aryl Sulfotransferase N

Hepatic aryl sulfotransferase IV was purified to apparent homogeneity from male Sprague-Dawley rats, using a modification4' of a published procedure.45 The enzyme was judged to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analytical isoelectric f o c ~ s i n g . ~ 'The , ~ ~subunit , ~ ~ relative molecular mass and the isoelectric point of the purified aryl sulfotransferase IV were identical to previously determined values.45 The specific activities of the homogeneous aryl sulfotransferase IV with 2naphthol as sulfuryl acceptor at pH 5.5 (780 nmol of p ro d u c t fo rme d lmin lmg p r o t e i n ) a n d w i t h 1naphthalenemethanol as sulfuryl acceptor at pH 7.0 (32 nmol product formed/min/mg protein) were similar to previously published values.42 Protein concentrations were determined49with bovine serum albumin as standard. Assay of Aryl Sulfotransferase N With Benzylic Alcohols

The various benzylic alcohols were evaluated as both substrates a n d inhibitors of purified a r y l sulfotransferase IV using a previously described HPLC method for determination of PAP formed in the r e a ~ tio n .~Reaction ' mixtures of 0.03 ml total volume contained 0.25 M potassium phosphate at pH 7.0, 8.3 mM 2-mercaptoethanol, 0.2 mM PAPS, and various concentrations of the benzylic alcohol in acetone (final concentration of acetone was 5% v/v). Reactions were initiated by addition of 1.0-1.5 k g enzyme, incubated at 37°C for 10 min, and then quenched by addition of an equal volume of methanol. After addition of methanol, each sample was thoroughly mixed and placed on ice MATERIALS AND METHODS until HPLC analysis. HPLC analysis of the reaction Enzgme Substrates and Assag Components mixture was carried out using a n Econosphere c18 colAll benzylic alcohols were obtained from Aldrich umn (250 mm x 4.6 mm, 5 km, Alltech) a t ambient Chemical Company. Optical purity was confirmed by temperature. The mobile phase consisted of waterpolarimetry with a Perkin Elmer Model 141 polarime- methanol (88:12) containing 50 mM potassium phoster. The specific rotations of the benzylic alcohols were phate, 50 m M ammonium chloride, and 1.0 m M 1as follows: ( - )-(1R,2S)-ephedrine, - 38.8" (in 1N HCI); octylamine; the pH of the mobile phase was adjusted to ( - )-(lR,2R)-pseudoephedrine, - 70.3" (in 1 N HCI); 5.45 before addition of methanol. The rate of the reaction was determined with a linear standard curve that ( + )-(lS,2R)-ephedrine hydrochloride, + 33.1" (in H,O); ( + )-(lS,2S)-pseudoephedrine hydrochloride, + 48.7" related HPLC peak area to the concentration of PAP formed in the reaction. The lower limit of detection in (in H,O); ( - )-(R)-1,2,3,4-tetrahydro-l-naphthol, - 33.6" (in CHC1,); ( + )-(S)-1,2,3,4-tetrahydro-l-these assays (at a signal-to-noise ratio of 7:l) correnaphthol, + 33.2" (in CHCI,); ( - )-(S)-2-methyl-l- sponded to a velocity of 0.6 nmol/min/mg of aryl sulfop h e n y l - 1 - p r o p a n o l , - 4 2 .8 " ( i n CHCl,); a n d transferase IV. + 41.2" (in For the determination of the kinetic constants for the ( + )-(R)-2-methyl-l-phenyl-l-propanol, CHCI,). Specific rotations were determined at 19°C for substrates, at least seven concentrations of the pertithe ephedrine and pseudoephedrine isomers, and at nent benzylic alcohol were assayed and these included 25°C for all others; all specific rotations were deter- concentrations both greater than and less than the apmined a t 589 nm. 3'-Phosphoadenosine-5'-phospho parent K,. For those benzylic alcohols that were inhibsulfate (PAPS) was prepared by chemical synthesis ac- itors of the enzyme, varying concentrations of the aland [35SlPAPSwas cohol were used to evaluate their inhibitory activity. cording to a published obtained from Dupont New England Nuclear. All other Initial rates (less than 10% of substrate used) were assay reagents and buffer components were obtained used for determination of kinetic constants. Apparent K , and V,, values were obtained by fitting the data from commercial sources.

106

RAO AND DUFFEL

to the Michaelis-Menten equation, V = V,, tSl/(K, + [SI), using the HYPER program described by Cleland.51Values for k,, were calculated using a relative molecular mass of 61,000 for aryl sulfotransferase IV, as previously estimated by gel f i l t r a t i ~ n Inhibition .~~ constants were obtained using the non-linear curve fitting program EZ-FIT described by Perrella.52

minicomputer. These minimum energy calculations are based on Allinger's MM2 molecular mechanics program and force-field parameters, as previously reviewed.54 RESULTS AND DISCUSSION

Stereoisomers of several chiral benzylic alcohols were examined for their ability to serve as substrates for aryl sulfotransferase IV. Kinetic constants for the Assay of Aryl Sulfotransferase Activity in Rat Hepatic enzyme-catalyzed reaction with each of the chiral ben100,OOOg Supernatant Fraction zylic alcohols are presented in Table 1. Catalytic effiA 100,OOOg supernatant fraction was prepared from ciency of the purified sulfotransferase was determined the combined livers of two male Sprague-Dawley by examination of the kcat/Kmvalues for each benzylic rats.42 Aryl sulfotransferase activity in the 100,OOOg alcohol. The enzyme displayed a stereochemical prefsupernatant fraction was determined using a mod- erence for the ( - )-(S)-enantiomer of 1-phenylethanol, i f i ~ a t i o nof~the ~ previously described thin-layer chro- and catalyzed the reaction with a 2-fold greater effimatographic assay for aryl sulfotransferase-catalyzed ciency than for the ( + )-(R)-enantiomer.This result was incorporation of 35S from [35S]PAPS.53These assays consistent with a previous report of a 3-fold difference were carried out at pH 7.0 and 37"C, with 0.05 mM in catalytic efficiency with the enantiomers of 11-naphthalenemethanol as substrate and varied con- p h e n y l e t h a n ~ l The . ~ ~ difference between 2-fold and 3c e n t r a t i o n s of ( + )-(R)-2-methyl-l-phenyl-l-fold enantioselectivity should not be considered signifpropanol or ( + )-(S)-1,2,3,4-tetrahydro-l-naphthol as icant in this case, due to statistical variability in the inhibitor. apparent K , and V,,, values for the enantiomers of 1-phenylethanol obtained with different preparations Partition Coefficientsof Benzylic Alcohols of the enzyme and different assay procedures. In the Partition coefficients were determined by allowing case of 2-methyl-l-phenyl-l-propanol, the purified aryl the various alcohols to partition between equal vol- sulfotransferase catalyzed stereospecific sulfation of umes of 0.25 M potassium phosphate in water at pH 7.0 the ( - )-(S)-enantiomer; the ( + )-(R)-enantiomer of 2(pre-saturated with 1-octanol) and 1-octanol (presatu- methyl-1-phenyl-1-propanol was not a substrate for the rated with the phosphate buffer). Alcohols were dis- enzyme. A similar absolute stereospecificity was obsolved in the aqueous phase at 0.4- 1.0 mg/ml and were served with the stereoisomers of ephedrine and pseuallowed to partition between the phases by shaking in doephedrine. That is, ( - )-(lR,lS)-ephedrine and sealed glass containers at 25°C for 72 h. The concen- ( - )-(lR,2R)-pseudoephedrinewere substrates for the tration of the benzylic alcohol in each phase was deter- aryl sulfotransferase, while ( + )-(lS,2R)-ephedrineand mined by ultraviolet absorbance. ( + )-(lS,2S)-pseudoephedrine were not substrates. Molecular modeling studies revealed that those benMolecular Graphics zylic alcohols that served as substrates for the enzyme Minimum energy conformations of the various ben- were similar in the orientation of the benzylic oxygen zylic alcohols were obtained using CHEM-X software and the larger alkyl substituent with respect to the (Chemical Design Ltd., Oxford) run on a VAX 11/780 plane of the phenyl ring. However, since compounds

TABLE 1. Summary of kinetic constants for sulfation of chiral benzylic alcohols catalyzed by purified aryl sulfotransferase IV

vm~=

(nmol/min/mg)

Benzylic alcohol 1 ( - )-(S)-1-Phenylethanol 2 ( +)-(R)-1-Phenylethanol 3 ( -)-(lR,2S)-Ephedrine 4 ( + )-(lS,2S)-Pseudoephedrine 5 ( - )-(lR,2R)-Pseudoephedrine 6 (+)-(lS,aR)-Ephedrine 7 ( - )-(S)-2-Methyl-l-phenyl-l-propanol 8 ( + )-(R)-2-Methyl-l-phenyl-l-propanol 9 ( - )-(R)-1,2,3,4-Tetrahydro-l-naphthol 10 ( + )-(S)-1,2,3,4-Tetrahydro-l-naphthol

*

0.36 0.04 0.602 0.05 6.99 k 0.93

11.0 k 3.2

-

1.362 0.22 -

0.03 k 0.003 -

22.0 k 0.4 18.85 0.3 22.5 5 1.6 0 40.4 8.2 0 6.1 5 0.3

*

0

28.7* 1.0 0

3.73 1.91 0.20

-

0.22

-

0.27 -

58.4

-

"Each value of apparent K , and V, is derived from initial velocities obtained at seven or more concentrationsof a benzylic alcohol. Standard errors of apparent K , and V, values indicate the precision of fitting these initial velocities and corresponding concentrations of benzylic alcohols to the Michaelis-Menten equation, as described in Materials and Methods.

STEREOSPECIFICITYOF ARYL SULFOTRANSFERASE IV

107

1-8 can assume many conformations due to rotation droxyl with respect to the phenyl ring and other subabout the carbon-carbon bond between the benzylic stituents on the benzylic carbon, the hydrophobic charcarbon a nd t he phenyl ring, 1,2,3,4-tetrahydro- acter of these benzylic alcohols contributed to their 1-naphthol was chosen as a model benzylic alcohol with ability to serve as substrates for the sulfotransferase. a more restricted range of conformations. As seen in Octanolhuffer partition coefficients (1-octanol/0.25 M (9) was potassium phosphate in water at pH 7.0) were deterTable 1, ( - )-(R)-1,2,3,4-tetrahydro-l-naphthol a n excellent substrate for aryl sulfotransferase IV, mined for compounds 1 (log P = 1.12),2 (log P = 1.131, while the ( + 1-6)-enantiomer (10) was not a substrate 3 (log P = - 0.48),5 (log P = - 0.471, 7 (log P = 1.28), for the enzyme. The catalytic efficiency, kCatlK,, of the and 9 (log P = 1.51). Comparison of these log P values enzyme with 9 (58.4 min-' mA-' was comparable to with the apparent K , values of the benzylic alcohol the value previously reported43 for l-naphthalene- substrates revealed that the K , values decreased in methanol (97 min-' d-'). relation to increasing logarithms of the corresponding Further molecular modeling studies revealed that octanolhuffer partition coefficients. Although these w h e n t h e c onf o rm atio n al e n e r g y of ( - )-(R)- data are limited in the number of log P values consid1,2,3,4-tetrahydro-l-naphthol was minimized, the re- ered, they are consistent with more extensive previous sulting torsion angle formed between the plane of the studies on the effect of hydrophobicity of other benzylic aromatic ring and the oxygen-benzylic carbon bond alcohols on their ability to serve as substrates for puwas - 62.89' (Figure 1).The conformational energy of rified aryl sulfotransferase IV.43 Therefore, the ability each of the other molecules (1-8) was then minimized, of chiral benzylic alcohols to serve as substrates for while keeping the torsion angle between the plane of aryl sulfotransferase IV is dependent on both the conthe aromatic ring and the oxygen-benzylic carbon bond figuration of the benzylic alcohol and the hydrophobicfixed a t - 63". As illustrated in Figure 2 for compounds ity of the molecule. 1,3, and 7, when the benzylic hydroxyl is at this angle, Since hydrophobicity is apparently a major contriball of the chiral benzylic alcohols having the alkyl sub- uting factor in the ability of benzylic alcohols to bind to stituent oriented on the opposite side of the plane of the aryl sulfotransferase IV, it would be expected that a aromatic ring were substrates for the sulfotransferase. change in configuration of the chiral benzylic carbon however, the would create a molecule with the same hydrophobicity, In the case of ( + )-(R)-l-phenylethano1(2), benzylic hydroxyl and the methyl group were not on yet without ability to serve a s substrate; the net effect opposite sides of the plane of the phenyl ring, yet 2 was would be to produce a n inhibitor of the enzyme. Indeed, still a substrate for the enzyme. As seen in Figure 2 , 2 a s s e e n i n F i g u r e 3 , ( + ) - ( S ) - 1 , 2 , 3 , 4 - t e t r a differs from compounds 4 , 6 , and 8 in that 2 can assume hydro-1-naphthol was a competitive inhibitor of aryl a conformation somewhat similar to those described for s u l f o t r a n s f e r a s e IV (Ki = 0.05 mM) w i t h 1compounds 1, 3, 5, and 7, with a minimum of steric naphthalenemethanol as substrate. Furthermore, was also a combulk on the same side of the phenyl ring as the benzylic ( + )-(R)-2-methyl-l-phenyl-l-propanol oxygen. This may explain the ability of ( + ) - ( R ) - petitive inhibitor of the enzyme (Ki = 2.1 mM). The 1-phenylethanol to serve as substrate for the enzyme, similarities between the Ki values for the inhibitors 8 and 10 and the apparent K , values for their respective although at a reduced catalytic efficiency. In addition to the orientation of the benzylic hy- enantiomers, 7 and 9, a s substrates indicate that over-

03 H OH

9

I

Q HO H

-

10

Fig. 1. Structural comparison of ( - )-(R)-1,2,3,4-tetrahydro-l-naphthol (9)and ( + )-(S)-1,2,3,4tetrahydro-1-naphthol(10).The molecular graphics representations of each enantiomer are calculated minimum energy conformations, with the aromatic rings of 9 and 10 displayed in the same relative position so as to indicate the angle between the plane of the aromatic ring and the oxygen-benzylic carbon bond. Each of these calculated structures is shown with the plane defined by the aromatic ring coincident with the z-axis and perpendicular to thez-y plane (i.e.,the plane of the paper);the asymmetric benzylic carbon is at the origin of the z,y,z coordinates, and the oxygen atom is labeled as 0.

108

RAO AND DUFFEL

n

3 -

7 -

Fig. 2. Structural comparison of ( - )-(S)-l-phenylethanol ( I ) , ( + )-(R)-1-phenylethanol (2), (-)-(lR,aS)-ephedrine (3),and ( - )-(S)-2-methyl-l-phenyl-l-propanol(7). All molecules are viewed with the plane defined by the phenyl ring coincident with the z-axis and perpendicular to the x-y plane (i,e., the plane of the paper); the benzylic carbon is at the origin of the x,yg coordinates. Oxygen and nitrogen atoms are labeled as 0 and N, respectively.

In addition to their ability to act as inhibitors of purified aryl sulfotransferase IV, compounds 8 and 10 also inhibited the formation of a sulfuric acid ester of 1-naphthalenemethanol catalyzed by the aryl sulfotransferase in a preparation of rat hepatic 100,OOOg supernatant fraction (Table 2). The extent of inhibition, however, was somewhat lower than that expected from studies with the purified enzyme. Factors such as binding of 8 and 10 to proteins other than aryl sulfotransferase IV in crude cytosolic preparations, and the possibility of binding to other sulfotransferases, might 0.01 4 0 10 20 30 40 50 60 result in low levels of inhibition. Although the possible interactions with other cytosolic proteins remain to be 1/[1 -Naphthalenernethanol] X lo3 CUM)-' determined, the data do indicate that compounds 8 and Fig. 3. Inhibition of purified aryl sulfotransferase IV by (+ )-(S)10 are inhibitors of aryl sulfotransferase in crude cy1,2,3,4-tetrahydro-l-naphthol (10). Data points are observed values, with the theoretical lines calculated for competitive i n h i b i t i ~ nCon. ~ ~ tosolic preparations. centrations of the inhibitor, 10, are represented as follows: no inhibA model for the active site of aryl sulfotransferase IV itor, closed circles; 0.03 mM, open squares; 0.08 mM, closed triangles; that incorporates the current data on chiral benzylic 0.13 mM, open circles; 0.18 mM, closed squares. alcohols is shown in Figure 4. The model includes a relatively nonspecific hydrophobic binding site for arall binding affinity at the active site of the enzyme is omatic groups, and a much more specific binding site not greatly affected by the configuration of the benzylic for PAPS. Although little is known about the enzymatcarbon. For those benzylic alcohols with a large sub- ic binding site and preferred conformational structure stituent on the benzylic carbon, however, the configu- of PAPS, it is important to note that PAPS is the only ration of the benzylic carbon determines whether the known sulfuryl donor for aryl sulfotransferase IVcatalyzed reactions, e.g., adenosine-5'-phosphosulfate compound acts as a substrate or an inhibitor.

STEREOSPECIFICITY OF ARYL SULFOTRANSFERASE IV

TABLE 2. Effect of chiral benzylic alcohol inhibitors on sulfotransferase activity in rat hepatic 100,OOOg supernatant fraction

109

aspect of stereospecificity is illustrated in the model by the ability of a bulky substituent, such as an isopropyl group, to fit in one configuration (as shown in Fig. 41, but steric constraints imposed by the enzyme surface Re1ativ e Inhibitor activity” prohibit free rotation of the bond between the benzylic carbon and the phenyl ring. The model shown in Figure 4 also incorporates preNoneb 100 vious studies on the kinetic and chemical mechanism of 0.05 mM ( + )-(S)-1,2,3,4-Tetrahydro-l-naphthol 86 aryl sulfotransferase IV with phenols as SO3 - accep0.25 mM ( + )-(S)-1,2,3,4-Tetrahydro-l-naphthol 73 91 1.0mM ( + )-(R)-2-Methyl-l-phenyl-l-propanol t o r ~ .These ~ ~ ,previous ~ ~ studies have determined that 3.0mM ( + )-(R)-2-Methyl-l-phenyl-l-propanol 84 the kinetic mechanism of the enzyme, a t pH 7.0 with 2-chloro-4-nitrophenolas substrate, is a random rapid “Sulfotransferaseactivity is expressed as a percentage relative to an equilibrium bi-bi mechanism with two dead-end prodassay without inhibitor.All rates are the mean of two determinations, ~ ~ ; was no stable enzymeand the maximum deviation from the mean for any single determi- uct inhibitor c o m p l e x e ~there bound SO3 intermediate detected. All kinetic data are nation was 2.1%. bThe control assay mixture contained 0.05 mM l-naphthalene- consistent with an enzymatic mechanism wherein a methanol as sulfiuyl acceptor, and other components as described in relatively nonpolar transition state exists during the the Materials and Methods section. The rate of product formation for transfer of the sulfuryl group from PAPS to an oxygen the control assay was 0.064 nmol/min/mg protein. atom on the other ~ubstrate.~’ The current studies with chiral benzylic alcohols have shown that there is also a will not serve as substrate for the enzyme.55Thus, as required stereochemical orientation of PAPS (sulfuryl depicted in Figure 4, there must be a specific binding donor) with respect to the sulfuryl acceptor. site for the 3’-phosphate on PAPS. The data on steIn conclusion, the stereochemistry of the benzylic reospecificity of the enzyme with chiral benzylic alco- carbon in chiral benzylic alcohols determines the type hols indicate that the stereochemistry of the orienta- of interaction seen with aryl sulfotransferase IV. In tion of the PAPS with respect to the benzylic hydroxyl molecules with a large bulky substituent on the benis also an important feature of the active site. Further- zylic carbon, one enantiomer is a substrate, while the more, there are apparently steric constraints in the other enantiomer is an inhibitor of the enzyme. These region of the benzylic hydroxyl that prevent conforma- insights into structure-activity relationships for tional rotation at the benzylic carbon in order to place biotransformation of benzylic alcohols in pathways inthe oxygen in the proper position for transfer of an volving sulfotransferases should be useful in prediction SO3- group from PAPS to the benzylic oxygen. This of detoxication as well as metabolic activation. Fur-

Fig. 4. Proposed model for the active site of aryl sulfotransferaseIV.The model illustrates the orias an example of an acceptorentation of PAPS with respect to (-)-(S)-2-methyl-l-phenyl-l-propanol substrate.

110

RAO AND DUFFEL

thermore, chiral benzylic alcohols such as ( + )-(S)1,2,3,4-tetrahydro-l-naphthol represent a new class of inhibitors for aryl sulfotransferase. Additional studies will be required, however, to determine the extent to which these results can be applied to other sulfotransferases, either in the rat or in other species. ACKNOWLEDGMENTS

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