Importance of peri-interactions on the stereospecificity of rat hydroxysteroid sulfotransferase STa with 1-arylethanols

Article abstract of DOI:10.1021/tx980219f

Hydroxysteroid (alcohol) sulfotransferases catalyze the sulfation of polycyclic aromatic hydrocarbons (PAHs) that contain benzylic hydroxyl functional groups. This metabolic reaction is often a critical step in the activation of a hydroxyalkyl-substituted PAH to form an electrophilic metabolite that is capable of forming covalent bonds at nucleophilic sites on DNA, RNA, and proteins. Since hydroxyalkyl-substituted PAHs are often metabolically formed by the stereoselective enzymatic hydroxylation of a benzylic position on an alkyl-substituted PAH, we have investigated the possibility that the sulfation of hydroxyalkyl aromatic hydrocarbons is also stereoselective. Homogeneous preparations of rat hepatic hydroxysteroid (alcohol) sulfotransferase STa were utilized to investigate the stereoselectivity of its catalytic function with the enantiomers of model 1- arylethanols. While only minimal stereoselectivity was observed for the catalytic efficiency of STa with the enantiomers of 1-(2-naphthyl)ethanol and 1-acenaphthenol, the enzyme was stereospecific for (R)-(+)-1-(1- naphthyl)ethanol, (R)-(+)-1-(1-pyrenyl)ethanol, and (R)-(+)-1-(9- phenanthryl)ethanol as substrates. Moreover, (S)-(-)-1-(1-naphthyl)ethanol, (S)-(-)-1-(1-pyrenyl)ethanol, and (S)-(-)-1-(9-phenanthryl)ethanol were competitive inhibitors of STa. Structural and conformational analyses of these 1-arylethanols indicated that steric interactions between the substituents on the benzylic carbon and the hydrogen in the peri-position on the aromatic ring system were important determinants of the stereospecificity of the enzyme with these molecules. The findings presented here have implications for the more accurate prediction of the ability of hydroxyalkyl- substituted PAHs to be activated via metabolic formation of electrophilic sulfuric acid esters.

Full text of DOI:10.1021/tx980219f

278
Chem. Res. Toxicol. 1999, 12, 278-285
Im p or ta n ce of per i-In ter a ction s on th e Ster eosp ecificity
of Ra t Hyd r oxyster oid Su lfotr a n sfer a se STa w ith
1-Ar yleth a n ols
Erden Banoglu and Michael W. Duffel*
Division of Medicinal and Natural Products Chemistry, College of Pharmacy,
The University of Iowa, Iowa City, Iowa 52242
Received September 25, 1998
Hydroxysteroid (alcohol) sulfotransferases catalyze the sulfation of polycyclic aromatic
hydrocarbons (PAHs) that contain benzylic hydroxyl functional groups. This metabolic reaction
is often a critical step in the activation of a hydroxyalkyl-substituted PAH to form an
electrophilic metabolite that is capable of forming covalent bonds at nucleophilic sites on DNA,
RNA, and proteins. Since hydroxyalkyl-substituted PAHs are often metabolically formed by
the stereoselective enzymatic hydroxylation of a benzylic position on an alkyl-substituted PAH,
we have investigated the possibility that the sulfation of hydroxyalkyl aromatic hydrocarbons
is also stereoselective. Homogeneous preparations of rat hepatic hydroxysteroid (alcohol)
sulfotransferase STa were utilized to investigate the stereoselectivity of its catalytic function
with the enantiomers of model 1-arylethanols. While only minimal stereoselectivity was
observed for the catalytic efficiency of STa with the enantiomers of 1-(2-naphthyl)ethanol and
1-acenaphthenol, the enzyme was stereospecific for (R)-(+)-1-(1-naphthyl)ethanol, (R)-(+)-1-
(1-pyrenyl)ethanol, and (R)-(+)-1-(9-phenanthryl)ethanol as substrates. Moreover, (S)-(-)-1-
(1-naphthyl)ethanol, (S)-(-)-1-(1-pyrenyl)ethanol, and (S)-(-)- 1-(9-phenanthryl)ethanol were
competitive inhibitors of STa. Structural and conformational analyses of these 1-arylethanols
indicated that steric interactions between the substituents on the benzylic carbon and the
hydrogen in the peri-position on the aromatic ring system were important determinants of
the stereospecificity of the enzyme with these molecules. The findings presented here have
implications for the more accurate prediction of the ability of hydroxyalkyl-substituted PAHs
to be activated via metabolic formation of electrophilic sulfuric acid esters.
Among xenobiotic alcohols that serve as substrates for
HSTs, benzylic alcohols derived from alkyl-substituted
polycyclic aromatic hydrocarbons (PAHs) are of signifi-
cant toxicological interest. Several of these compounds
are metabolized to electrophilic sulfuric acid esters that
are capable of forming covalent DNA adducts (10-12).
Direct evidence for the metabolic formation of electro-
philic sulfuric acid esters from hydroxymethyl PAHs was
reported by Watabe and co-workers (13), when they
isolated an unstable sulfuric acid ester of 7-(hydroxy-
methyl)-12-methylbenz[a]anthracene (HMBA) from rat
liver cytosol that had been fortified with 3′-phospho-
adenosine 5′-phosphosulfate (PAPS), the physiological
cosubstrate for sulfotransferases. Furthermore, the sul-
furic acid ester of HMBA was shown to be strongly
mutagenic and found to form covalent bonds with the
amino groups of purine bases in calf thymus DNA (14).
Other examples of hydroxymethyl PAHs that are meta-
bolically activated via sulfuric acid esterification include
1-(hydroxymethyl)pyrene (15), 5-(hydroxymethyl)chry-
sene (16), 6-(hydroxymethyl)benzo[a]pyrene (17), and
9-(hydroxymethyl)-10-methylantharacene (18). Sulfation
of these hydroxymethyl PAHs was significantly inhibited
by DHEA, a typical substrate for hydroxysteroid (alcohol)
sulfotransferases (12, 17-19).
In tr od u ction
Hydroxysteroid (alcohol) sulfotransferases (HSTs)1 cata-
lyze the formation of sulfuric acid esters from a wide
range of endogeneous and xenobiotic alcohols as well as
from neutral hydroxysteroids such as dehydroepiandro-
sterone (DHEA) (1-4). Those sulfotransferases that
can catalyze the sulfation of xenobiotic alcohols and
hydroxysteroids have been organized into gene families,
and various systems of nomenclature have been described
elsewhere (5-7). In addition to these systems of nomen-
clature, the Human Genome Organization now recog-
nizes SULT as a prefix for systematic classification of
human sulfotransferases. This report involves the major
hepatic sulfotransferase in female rats which is most
often identified by the trivial name hydroxysteroid sul-
fotransferase a (STa) as assigned in the purification
described by Watabe and co-workers (8, 9).
* To whom correspondence should be addressed. Telephone: (319)
335-8840. Fax: (319) 353-5594. E-mail: michael-duffel@uiowa.edu.
1
Abbreviations: DHEA, dehydroepiandrosterone; DIP-chloride, B-
chlorodiisopinocampheylborane; HST, hydroxysteroid (alcohol) sulfo-
transferase; kcat, catalytic turnover number (moles of sulfuric acid ester
product formed per minute per mole of STa subunit); log P, logarithm
of the partition coefficient; MTPA, R-methoxy-R-(trifluoromethyl)-
phenylacetic acid; NOE, nuclear Overhauser effect; PAH, polycyclic
aromatic hydrocarbon; PAP, adenosine 3′,5′-diphosphate; PAPS, 3′-
phosphoadenosine 5′-phosphosulfate; ROESY, rotating frame nuclear
Overhauser effect NMR spectrometry; SDS-PAGE, sodium dodecyl
sulfate-polyacrylamide gel electrophoresis; STa, rat hydroxysteroid
(alcohol) sulfotransferase a.
In addition to the hydroxymethyl PAHs that are
converted to electrophilic metabolites through sulfation,
a few PAHs containing secondary benzylic hydroxyl
10.1021/tx980219f CCC: $18.00 © 1999 American Chemical Society
Published on Web 02/12/1999

Stereospecificity of Sulfotransferase STa
Chem. Res. Toxicol., Vol. 12, No. 3, 1999 279
Syn th esis of 1-Acen a p h th en on e. Acenaphthene (1.54 g in
20 mL of acetic acid) was treated at 50 °C with 3 g of chromium
trioxide in 3 mL of H2O and 8 mL of acetic acid. The mixture
was stirred at this temperature for 1 h and poured into water
to yield crude 1-acenaphthenone. The crude product was purified
by flash chromatography (8:2 hexane/ethyl acetate) and recrys-
tallized from an ethanol/water mixture. The resulting yellow
crystals had a melting point of 119-120.5 °C [lit. (25) mp 120.5
°C]. The chemical structure of 1-acenaphthenone was verified
by IR and 1H NMR spectroscopy, and the spectral data were
identical with values for 1-acenaphthenone that were previously
reported (25).
group(s) have also been shown to be metabolically
activated by sulfotransferases. For example, both 4-hy-
droxy-3,4-dihydrocyclopenta[cd]pyrene and 3,4-dihydroxy-
3,4-dihydrocyclopenta[cd]pyrene, major metabolites of
the environmental carcinogen cyclopenta[cd]pyrene, were
converted to electrophilic sulfuric acid esters in sulfo-
transferase-catalyzed reactions (20). 1-(1-Pyrenyl)etha-
nol, 1-(2-pyrenyl)ethanol, 10H-indeno[1,2,7,7a-bcd]pyren-
10-ol, and 1-(6-benzo[a]pyrenyl)ethanol were metabolically
activated by PAPS-fortified cytosolic fractions derived
from the livers of female rats, and the level of sulfation
of these PAH derivatives correlated with the greater
hepatic activity of HSTs in female rats as opposed to male
rats (15, 21). With the exception of 1-(1-pyrenyl)ethanol,
all secondary benzylic alcohols in these studies were used
as racemates, and no indication of stereoselectivity was
reported in these metabolic activations.
STa has been purified from hepatic cytosol derived
from female Sprague-Dawley rats and shown to exhibit
high catalytic activity for hydroxymethyl PAHs as well
as for hydroxysteroids such as DHEA (8, 9). This work
clearly showed that homogeneous STa catalyzed the
sulfation of four hydroxymethyl PAHs. However, since
the relative activities of the enzyme with these hy-
droxymethyl PAHs were determined at a single concen-
tration of substrate, the effect of structure on the catalytic
efficiency of the STa with this class of substrates was
not addressed.
Syn th esis of Ra cem ic 1-Ar yleth a n ols. Racemates of 1-(1-
pyrenyl)ethanol and 1-(9-phenanthryl)ethanol were synthesized
by the sodium borohydride reduction of 1-acetylpyrene and
9-acetylphenanthrene, respectively. To a solution of the ap-
propriate ketone (1.2 mmol) in 20 mL of ethanol was added
sodium borohydride (12 mmol). The reaction mixture was stirred
at room temperature for 2 h. After the reaction mixture was
evaporated to dryness, water was added to decompose the excess
sodium borohydride. The solution was then extracted with ethyl
acetate (2 × 50 mL). The combined organic layers were
evaporated to yield the crude 1-arylethanol, which was subse-
quently purified by flash column chromatography with hexane/
ethyl acetate (8:2) as the mobile phase. Yields of 91-96% were
obtained for each of the two racemic 1-arylethanols. Chemical
structures of the product racemic 1-arylethanols obtained as
products were verified by 1H NMR spectroscopy, IR spectros-
copy, and GC/MS, and these data were identical with literature
values reported for the racemic compounds (26).
Syn th esis of Ch ir a l 1-Ar yleth a n ols. Optically active (R)-
(+)- and (S)-(-)-1-arylethanols were synthesized by asymmetric
reduction of the appropriate ketone with either (+)- or (-)-DIP-
chloride, respectively, according to a previously published
general procedure (27). All glassware was oven-dried overnight,
and all operations were carried out under an argon atmosphere.
To a solution of 2.97 mmol of (+)- or (-)-DIP-chloride in
tetrahydrofuran (5 mL) at -25 °C was added the appropriate
ketone (2.97 mmol). A yellow color developed immediately. The
reaction was carried out at this temperature for 11 h. The
R-pinene was removed under reduced pressure (0.1 mmHg)
overnight. The residue was dissolved in diethyl ether (50 mL),
and diethanolamine (2.2 equiv) was added. The separated solid
was filtered off after 2 h and washed twice with n-pentane (30
mL). The combined diethyl ether and n-pentane filtrates were
concentrated, and the product chiral alcohol was purified by
flash column chromatography with hexane/ethyl acetate (9:1)
as the mobile phase. Yields of 50-75% were obtained for each
of the six chiral 1-arylethanols prepared with this procedure.
The IR, NMR, and GC/MS spectral data for the optically active
alcohols were identical with those of authentic racemic materials
(26).
Previously, we have reported quantitative structure-
activity studies on the hydrophobic and steric require-
ments necessary for the optimal catalytic efficiency of STa
with a series of model primary benzylic alcohols (22). We
also have observed stereoselectivity in the catalytic
efficiency of STa with a series of R-alkyl-substituted
chiral secondary benzylic alcohols (23). In the study
described here, we have utilized enantiomerically pure
stereoisomers of 1-arylethanols as model compounds to
determine the stereochemical requirements for participa-
tion of STa in the sulfation of hydroxyalkyl PAHs.
Ma ter ia ls a n d Meth od s
Analysis by thin-layer chromatography (TLC) was performed
on precoated, 250 µm thick, silica gel GF plates (Analtech,
Newark, DE). Purifications by flash chromatography were
carried out on silica gel (200-400 mesh, 60 Å) from Aldrich
Chemical Co. (Milwaukee, WI). Stereoisomers of 1-(1-naphthyl)-
ethanols, 1-(2-naphthyl)ethanols, (()-1-acenaphthenol, 1-acetyl-
pyrene, 9-acetylphenanthrene, and acenaphthene were obtained
from Aldrich, and their chemical purity was confirmed by TLC.
Optical purities of the benzylic alcohols were determined with
a Perkin-Elmer 141 polarimeter. The specific rotations of the
benzylic alcohols were as follows: (R)-(+)-1-(1-naphthyl)ethanol,
+75.6° (c 0.01, CH3OH); (S)-(-)-1-(1-naphthyl)ethanol, -74.8°
(c 0.01, CH3OH); (R)-(+)-1-(2-naphthyl)ethanol, +36.2° (c 0.015,
C2H5OH); and (S)-(-)-1-(2-naphthyl)ethanol, -37.7° (c 0.015,
C2H5OH). Specific rotations were determined at 589 nm and at
25 °C for all benzylic alcohols. The (+)- and (-)-isomers of DIP-
chloride (B-chlorodiisopinocampheylborane) were used as ob-
tained from Aldrich. 3′-PAPS was prepared according to a
published procedure (24). All other assay components and
reagents were obtained from commercial sources and were of
the highest purity available. Chemical structures of the product
1-arylethanols were verified by 1H NMR spectroscopy (Bruker
WM-360), IR spectroscopy (Nicolet model 205), and GC/MS
(Hewlett-Packard 5989 quadruple GC/MS system). Ca u tion :
Polycyclic aromatic hydrocarbons and derivatives such as the
polycyclic 1-arylethanols should be handled with appropriate
safety precautions to avoid exposure.
P r ep a r a tion of r-Meth oxy-r-(tr iflu or om eth yl)p h en yl-
a cetic Acid (MTP A) Ester s of Ra cem ic a n d Ch ir a l 1-Ar yl-
eth a n ols. MTPA esters of enantiomers of product alcohols (both
chiral and racemic) were prepared by mixing the appropriate
alcohol (1 mmol), (S)-MTPA (1 mmol), dicyclohexylcarbodiimide
(1 mmol), and 4-(dimethylamino)pyridine (0.1 mmol) in meth-
ylene chloride (10 mL) at room temperature overnight. The
dicyclohexylurea that precipitated was removed by filtration.
The eluate was washed successively with 50 mL portions of 0.5
N HCl, 2 N Na2CO3, and brine. The crude product obtained after
evaporation of the organic layer was purified by flash column
chromatography with hexane/ethyl acetate (8:2) as the mobile
phase. The 1H NMR spectrum of the MTPA ester of each racemic
benzylic alcohol displayed two sets of signals due to the methoxy
and benzylic methine protons, whereas each enantiomerically
pure benzylic alcohol showed only a single set of signals.
1-Acen a p h th en ols. Stereoisomers of 1-acenaphthenol were
prepared from 1-acenaphthenone.
For (R)-(-)-1-acenaphthenol, the ee was determined to be
>99% from the 1H NMR spectrum of MTPA ester: mp 126-

280 Chem. Res. Toxicol., Vol. 12, No. 3, 1999
Banoglu and Duffel
127 °C [lit. (28) 127-127.5 °C]; [R]25 -1.5° (c 0.06, CHCl3) [lit.
D
(28) -1.46° (c 1.23, CHCl3)].
For (S)-(+)-1-acenaphthenol, the ee was determined to be
>99% from the 1H NMR spectrum of MTPA ester: mp 126-
127 °C; [R]25 +1.41° (c 0.06, CHCl3) [lit. (28) ee 88%, +1.34° (c
D
1.16, CHCl3)].
1-(1-P yr en yl)eth a n ols. Stereoisomers of 1-(1-pyrenyl)etha-
nol were prepared from 1-acetylpyrene.
For (R)-(+)-1-(1-pyrenyl)ethanol, the ee was determined to
1
be 98% from the H NMR spectrum of MTPA ester: mp 95-96
°C; [R]25 +53.2° (c 0.13, CHCl3) [lit. (26) ee 80%, +44° (c 0.9,
D
CHCl3)].
For (S)-(-)-1-(1-pyrenyl)ethanol, the ee was determined to be
1
95% from the H NMR spectrum of MTPA ester: mp 96-98 °C;
[R]25 -55° (c 0.06, CHCl3).
D
F igu r e 1. Structures of arylethanols investigated as substrates
1-(9-P h en a n th r yl)eth a n ols. Stereoisomers of 1-(9-phenan-
thryl)ethanol were prepared from 9-acetylphenanthrene.
For (R)-(+)-1-(9-phenanthryl)ethanol, the ee was determined
and inhibitors of STa.
St. Louis, MO) on an Indigo workstation (Silicon Graphics,
Mountain View, CA). Energy minimization for each molecule
was performed using Maximin2 with the Tripos force field.
Ca lcu la tion of Hyd r op h obicity Con sta n ts for 1-Ar yleth -
a n ols. Partition coefficients for 1-arylethanols were calculated
using the ACD/LogP software from Advanced Chemistry De-
velopment Inc.
1
to be 96% from the H NMR spectrum of MTPA ester: mp 125-
127 °C; [R]25 +71.2° (c 0.04, CHCl3) [lit. (26) ee 87%, +39.9° (c
D
0.6, CHCl3)].
For (S)-(-)-1-(9-phenanthryl)ethanol, the ee was determined
1
to be 99% from the H NMR spectrum of MTPA ester: mp 125-
127 °C; [R]25 -73.4° (c 0.03, CHCl3).
D
NMR. One-dimensional NOE spectra of (S)-(-)-1-(1-naphth-
yl)ethanol were obtained using a Bruker WM-360 MHz spec-
trometer. The two-dimensional ROESY spectra were obtained
using a Varian UNITY 500 pulsed Fourier transform spectrom-
eter. Four transients were averaged to produce each 2048-point
FID, with 330 complex traces collected in the F1 dimension.
Phase cycling for the F1 dimension used the hypercomplex
method (29). The relaxation delay was set to 6 s, and mixing
times ranged from 0.025 to 0.75 s. F2 processing was performed
with a 2048-point Fourier transformation apodized by a 0.079
s Gaussian function. F1 data were zero filled to 1024 points,
followed by transformation under a 0.025 s Gaussian function.
Spline baseline correction was applied in the F2 dimension
before measurement of peak intensities. Buildup curves were
generated by monitoring the cross-peak intensity and volume
as a function of mixing time. All data collection, processing, and
analysis were performed using the Varian VNMR 4.3B package.
For the calculation of the relative interatomic distances, the
rotating frame Overhauser enhancements measured from ROE-
SY cross-peak volumes (30) were normalized to the volume of
the origin diagonal peak at each mixing time.
X-r a y Cr ysta llogr a p h y. Data for (S)-(-)-1-(1-naphthyl)-
ethanol (a colorless thin rectangular lathe, 0.08 mm × 0.24 mm
× 0.62 mm) and (S)-(-)-1-(1-pyrenyl)ethanol (a colorless prism,
0.18 mm × 0.13 mm × 0.088 mm) were collected on an Enraf-
Nonius CAD4 diffractometer (MoKR radiation, graphite mono-
chromator) using θ-2θ scans. Intensity standards were mea-
sured at 2 h intervals. Net intensities were obtained by profile
analysis of the data. Lorentz and polarization corrections were
applied. The computer programs from the MoLEN package were
used for data reduction.
Assa y of H yd r oxyst er oid Su lfot r a n sfer a se STa w it h
1-Ar yleth a n ols. The model 1-arylethanols were evaluated as
both substrates and inhibitors of purified STa using a published
HPLC procedure for determination of PAP formed in the
reaction (32). Reaction mixtures with a 0.03 mL total volume
contained 0.25 M potassium phosphate buffer (pH 7), 8.3 mM
2-mercaptoethanol, 0.3 mM PAPS, and various concentrations
of the alcohols in acetone (final concentration of acetone in the
assay was no more than 5% v/v). Reactions were initiated by
addition of 1.0-3.0 µg of enzyme; the mixtures were incubated
at 37 °C for 10-30 min, and the reactions were terminated by
addition of 0.03 mL of methanol. The concentration of PAP
formed in the reaction was determined by HPLC. Linear
standard curves relating HPLC peak areas to concentrations
of PAP were determined daily. At least six different concentra-
tions of each alcohol were assayed, and these included concen-
trations both greater than and less than the apparent Km.
Apparent Km and Vmax values are presented as (the standard
error obtained by nonlinear least-squares curve fitting (33) of
the velocity data to the Michaelis-Menten equation. Values for
kcat were calculated using a relative molecular mass of 33 124
Da for a subunit of STa as determined from the deduced amino
acid sequence (34). Approximate values for Km and Vmax with
(R)-(+)-1-(1-pyrenyl)ethanol were obtained by analyzing reaction
velocities obtained in the presence of the alternative substrate
p-butylbenzylalcohol as described by the equation
vt ) (VmaxA[A]/KmA + VmaxB[B]/KmB)/
(1 + [A]/KmA + [B]/KmB
)
where VmaxA and VmaxB are the maximal velocities with A and
B as substrates, respectively (35). The observed velocity (vt) of
the reaction for each mixed concentration of both substrates was
determined by varying the concentrations of both p-butylben-
zylalcohol and (R)-(+)-1-(1-pyrenyl)ethanol. As a result, the
kinetic constants KmB and VmaxB for (R)-(+)-1-(1-pyrenyl)ethanol
were obtained by nonlinear least-squares curve fitting of the
observed velocity data to the above equation.
A preliminary model of the structure was obtained using
MULTAN, a direct methods program. Least-squares refining
of the model versus the data was performed with the XL
computer program of the SHELXTL v5.0 package. Illustrations
were made with the XP program, and tables were made with
the XCIF program. Both are in the SHELXTL package. Thermal
ellipsoids shown in the illustrations are at the 35% level.
P u r ifica tion of Hyd r oxyster oid Su lfotr a n sfer a se STa .
Hydroxysteroid sulfotransferase STa was purified to homogene-
ity from female Sprague-Dawley rats, using a modification (22)
of previously published procedures (8, 9). The enzyme was
homogeneous as determined by SDS-PAGE with Coomassie
Blue staining. Protein concentrations were determined using a
modified Lowry procedure (31) with bovine serum albumin as
the standard.
Resu lts
Ca ta lytic Efficien cy of STa w ith 1-Ar yleth a n ols.
Stereoisomers of several chiral 1-arylethanols (Figure 1)
were examined for their ability to interact with hydroxy-
steroid (alcohol) sulfotransferase STa. Kinetic constants
for each STa-catalyzed sulfation reaction are presented
in Table 1. The difference between the interaction of STa
Molecu la r Mod elin g. Molecular models of 1-arylethanols
were built using the Sybyl software package (Tripos Associates,

Stereospecificity of Sulfotransferase STa
Chem. Res. Toxicol., Vol. 12, No. 3, 1999 281
Ta ble 1. Su m m a r y of Kin etic Con sta n ts for STa -Ca ta lyzed Su lfa tion of 1-Ar yleth a n olsa
1-arylethanol
Km
Vmax
kcat/Km
Ki
(R)-(+)-1-(1-naphthyl)ethanol
(S)-(-)-1-(1-naphthyl)ethanol
(R)-(+)-1-(9-phenanthryl)ethanol
(S)-(-)-1-(9-phenanthryl)ethanol
(R)-(+)-1-(1-pyrenyl)ethanol
(S)-(-)-1-(1-pyrenyl)ethanol
(R)-(-)-1-acenaphthenol
800 ( 100
-
52.0 ( 2.9
-
2.2 ( 0.3
-
-
250 ( 20
90 ( 20
-
4.5 ( 0.5
-
1.7 ( 0.4
-
-
10 ( 1
7 ( 0.6
-
560 ( 60
400 ( 40
1000 ( 170
1200 ( 200
11.0 ( 1.0
-
83.0 ( 3.3
121.0 ( 3.7
34.0 ( 2.2
28.5 ( 1.8
49.2 ( 6.0
-
4.8 ( 0.5
9.7 ( 0.9
1.1 ( 0.19
0.8 ( 0.13
-
2 ( 0.2
-
-
-
-
(S)-(+)-1-acenaphthenol
(R)-(+)-1-(2-naphthyl)ethanol
(S)-(-)-1-(2-naphthyl)ethanol
a
Values for apparent Km, Vmax, kcat/Km, and Ki are expressed in µM, nmol min-1 (mg of STa)-1, min-1 mM-1, and µM, respectively.
The Ki value for (S)-(-)-(1-naphthyl)ethanol was determined with (R)-(+)-(1-naphthyl)ethanol as the substrate, and the Ki values for
(S)-(-)-1-(9-phenanthryl)ethanol and (S)-(-)-1-(1-pyrenyl)ethanol were determined with 4-butylbenzylalcohol as the substrate.
with the enantiomers of 1-substituted and 2-substituted
naphthalene compounds was substantial. In the case of
1-(2-naphthyl)ethanol, STa catalyzed the sulfation of both
enantiomers, but the effect of stereochemistry on the
catalytic efficiency of STa was small, as indicated by a
less than 2-fold preference for the (R)-(+)-enantiomer.
However, with 1-(1-naphthyl)ethanol, STa clearly showed
stereospecificity. As seen from Table 1, (R)-(+)-1-(1-
naphthyl)ethanol was a substrate for the enzyme, while
the (S)-(-)-enantiomer was a competitive inhibitor of the
sulfation of the (R)-(+)-enantiomer catalyzed by STa.
The stereospecificity exhibited with 1-(1-naphthyl)-
F igu r e 2. Conformations of (S)-(-)-1-(1-naphthyl)ethanol ob-
served by X-ray diffraction of a single crystal. Hydrogen bonding
between two molecules of (S)-(-)-1-(1-naphthyl)ethanol with
differing conformations is shown (labeled as O11-H-O31).
ethanol prompted the examination of other 1-aryletha-
nols. A stereospecificity similar to that seen with 1-(1-
naphthyl)ethanol was observed with the stereoisomers
of 1-(1-pyrenyl)ethanol and 1-(9-phenanthryl)ethanol. In
both cases, the (R)-(+)-enantiomers were substrates,
while the (S)-(-)-isomers were not substrates for STa.
However, (S)-(-)-1-(1-pyrenyl)ethanol and (S)-(-)-1-(9-
phenanthryl)ethanol were competitive inhibitors of the
enzyme. In the case of 1-acenaphthenol, a molecule where
the conformational changes of the benzylic alcohol moiety
were restricted by fixing it into the ring system, both
enantiomers were substrates for STa, and STa catalyzed
the sulfation of the (S)-(+)-enantiomer with a 2-fold
difference in the catalytic efficiency.
In the case of (R)-(+)-1-(1-pyrenyl)ethanol, although it
was shown to be a substrate for STa, it was not
experimentally possible to calculate accurate Km and Vmax
values due to limitations on the reliable determination
of the concentrations of PAP formed at the lowest
concentrations of substrate required. Therefore, we used
an alternative approach to determine the kinetic con-
stants for this substrate. As outlined in Materials and
Methods, we utilized a kinetic analysis of the velocity of
the sulfation reaction under conditions where two sub-
strates compete for the same enzyme (35). As seen in
Table 1, the use of p-butylbenzylalcohol as a competing
substrate provided data that yielded an estimate of 7 µM
as the apparent Km for (R)-(+)-1-(1-pyrenyl)ethanol, with
a maximal velocity of 11 nmol of product formed min-1
(mg of STa)-1. The (S)-(-)-isomer was an inhibitor of STa
with a Ki of 2 µM. Within the limits of detection of the
assay method (i.e., 0.3 nmol of product formed min-1
mg-1), (S)-(-)-1-(1-pyrenyl)ethanol did not serve as a
substrate for STa.
the (S)-(-)-1-(1-naphthyl)ethanol molecules appeared in
two different conformations that were hydrogen bonded
to each other in a single crystal. The only significant
difference between the two conformations was in the
conformation of the O11 (O31) hydroxyl groups relative
to the plane of the naphthalene ring. Comparison of the
corresponding torsion angles (i.e., C2-C1-C11-O11 )
-27° vs C22-C21-C31-O31 ) -43°) indicated the O31
group was rotated 16° from the conformation of the O11
group. This minor difference was probably an accom-
modation to the hydrogen bonding chains. In the case of
the (S)-(-)-1-(1-pyrenyl)ethanol, the molecule appeared
in four different conformations within a single crystal
(Figure 3). Three of these conformations (conformations
A-C in Figure 3) were in fact very similar, and the
differences in the corresponding torsion angles for the
O19A, O19B, and O19C groups relative to the plane of
pyrene ring (i.e., C2A-C1A-C17A-O19A ) -18.7°,
C2B-C1B-C17B-O19B ) -17.8°, and C2C-C1C-
C17C-O19C ) -8.0°) were very small. However, the
fourth conformation (conformation D in Figure 3) sub-
stantially differed from the other three with respect to
the conformation of O19D. The corresponding torsion
angle for the O19D group (C2D-C1D-C17D-O19D )
-111°) indicated that the O19D group was rotated about
90-100° from the conformations of other hydroxyl groups.
In addition, evidence for the conformation of (S)-(-)-
1-(1-naphthyl)ethanol in solution was obtained by one-
dimensional differential NOE and two-dimensional ROE-
SY NMR in chloroform-d. An experiment in which one-
dimensional differential NOE NMR clearly displayed
through-space proximity of the peri-hydrogen (H at the
8-position on naphthalene ring) and CH of the hy-
droxymethyl substituent at the 1-position (data not
shown), suggesting that the preferred conformation in
Con for m a t ion a l An a lysis of (S)-(-)-1-(1-Na p h -
th yl)eth a n ol a n d (S)-(-)-1-(1-P yr en yl)eth a n ol. Rela-
tive conformations of (S)-(-)-1-(1-naphthyl)ethanol and
(S)-(-)-1-(1-pyrenyl)ethanol were determined by X-ray
crystallography (Figures 2 and 3). As shown in Figure 2,

282 Chem. Res. Toxicol., Vol. 12, No. 3, 1999
Banoglu and Duffel
F igu r e 3. Conformations of (S)-(-)-1-(1-pyrenyl)ethanol observed by X-ray diffraction. The four conformations observed in a single
crystal are shown.
the liquid was similar to the conformation in the crystal
state. This was further supported by calculating the
relative interatomic distances between the peri-hydrogen
and substituents at the 1-position by ROESY (30). The
calculated relative distances were 2.2 ( 0.2 Å for CH-
H8 and 3.25 ( 0.11 Å for CH3-H8. These relative
distances were similar to those calculated from the X-ray
structure: CH-H8, 2.24 Å; CH3-H8, 3.4 Å.
state. Therefore, our modeling studies revealed that,
among the molecules that were inhibitors of STa, there
were similarities in the orientations of the alcohol
functionality and the methyl substituent with respect to
the plane of the aromatic ring. Although 1-(2-naphthyl)-
ethanol may assume many conformations due to possible
rotation about the carbon-carbon bond between the
benzylic carbon and the naphthyl ring, a similar rotation
was restricted for 1-(1-naphthyl)-, 1-(1-pyrenyl)-, and
1-(9-phenanthryl)ethanol (Figure 4).
The relative position of the OH moiety with respect to
the aromatic ring and the peri-hydrogen can be defined
by the torsion angle ω (Ã11-C11-C1-C2); this torsion
angle is -27° in the crystal state (Figure 2). The change
in this torsion angle defines the rotation about the
carbon-carbon bond between the benzylic carbon (C11)
and the aromatic carbon (C1) and, thus, can be used to
determine the conformers of 1-(1-naphthyl)ethanol at
different energies by using molecular modeling tools.
Figure 4 shows the changes in conformational energy as
a function of torsion angle ω. The angles were measured
by a clockwise rotation of an oxygen atom (O11) with
respect to aromatic carbon atom C2. When O11 eclipsed
C2 (O11, C11, C1, and C2 lie in the same plane), the
torsion angle was defined as 0° for the purpose of the
modeling experiments.
Molecu la r Mod elin g Stu d ies. Molecular modeling
studies using force field calculations further revealed that
conformation (a) of (S)-(-)-1-(1-naphthyl)ethanol deter-
mined from X-ray (Figure 2) and NMR studies was in
fact the lowest-energy conformation. When the confor-
mational energy of (S)-(-)-1-(1-naphthyl)ethanol was
minimized, the resulting torsion angle ω (C2-C1-C11-
O11) formed between the plane of the aromatic ring and
the oxygen of the alcohol moiety was about -27°, as
observed in the crystal state (Figure 2). Therefore, we
chose this torsion angle as a starting point for investigat-
ing the similarities among these 1-arylethanols. The
conformational energies of (S)-(-)-1-(1-pyrenyl)ethanol
and (S)-(-)-1-(9-phenanthryl)ethanol were then mini-
mized while keeping the torsion angle between the plane
of the aromatic ring and the benzylic hydroxyl fixed at
-27°. When the benzylic alcohol was at this angle, the
methyl substituent was always oriented at the opposite
side of the aromatic ring plane with a torsion angle of
about 90° between the methyl carbon and aromatic ring.
The benzylic hydroxyl assumed a conformation anti from
the peri-hydrogen (H8) below the naphthalene ring, and
this was very similar to what was observed in the crystal
As shown in Figure 4, the calculated differences in the
energy between the conformers of (S)-(-)-1-(1-naphthyl)-
ethanol, (S)-(-)-1-(1-pyrenyl)ethanol, and (S)-(-)-1-(9-
phenanthryl)ethanol are much higher than the energy
difference between the conformers of (S)-(-)-1-(2-naph-

Stereospecificity of Sulfotransferase STa
Chem. Res. Toxicol., Vol. 12, No. 3, 1999 283
isoform of sulfotransferase catalyzing sulfation of hy-
droxyalkyl PAHs (9), and a series of chiral 1-arylethanols
beginning with 1-(1-naphthyl)ethanol. In naphthalene,
the 1- and 8-positions are said to be peri to each other.
In view of the geometry of naphthalene, substituents
located at these positions are in much closer proximity
than similar substituents located ortho to each other, and
steric interactions should be more severe. This closer
proximity has been responsible for the appearance of
several unique properties of peri-substituted naphtha-
lenes that have been reported by different groups over
many years (39-44).
Our results with 1-(1-naphthyl)-, 1-(1-pyrenyl)-, and
1-(9-phenanthryl)ethanol show that conformational
changes of the benzylic hydroxyl in these molecules are
restricted to a certain range of torsion angles due to steric
interactions between the peri-hydrogen (H8) and the
methyl and hydroxyl groups of the hydroxymethyl sub-
stituent at the 1-position on naphthalene and pyrene, and
the corresponding 9-position on phenanthrene. As shown
in Figure 4, examination of the conformational changes
of the benzylic hydroxyl group as a function of rotation
about the C-C bond between the benzylic carbon and
the aromatic ring carbon indicates that the differences
in energy between these 1-arylethanols and 1-(2-naph-
thyl)ethanol are greater when the methyl and hydroxyl
groups are closest to the peri-hydrogen. This effectively
means that conformational changes of the benzylic
alcohol moiety of these 1-arylethanols with peri-substitu-
ent interactions are restricted to a limited range of
torsion angles. These results also correlate well with the
torsion angles that were obtained from the crystal
conformations of these molecules (Figures 2 and 3) and
data obtained from two-dimensional ROESY NMR analy-
sis of (S)-(-)-1-(1-naphthyl)ethanol. While it is recognized
that molecular conformations within crystals may depend
on solvent and crystallization conditions, our data for
single crystals of 1-(1-naphthyl)ethanol and 1-(1-pyrenyl)-
ethanol, each under a single crystallization condition, are
consistent with the predictions obtained from our mo-
lecular modeling studies with molecules with peri-
interactions.
In the case of the (S)-(-)-1-(2-naphthyl)ethanol, how-
ever, peri-substituent interactions are not present, and
the benzylic hydroxyl may assume many conformations
with a minimum of steric interactions between the
methyl group and ortho hydrogens. Some of these con-
formations are very likely suitable for sulfuryl group
transfer from PAPS at the active site, and this may
explain the ability of the (S)-(-)-1-(2-naphthyl)ethanol
to serve as a substrate for the enzyme, although at a
reduced catalytic efficiency. Therefore, it can be assumed
that in the case of the (S)-(-)-enantiomers of 1-(1-
naphthyl)-, 1-(1- pyrenyl)-, and 1-(9-phenanthryl)ethanol
where there are peri-substituent interactions, the pre-
ferred configuration would be the one that is unproduc-
tive with respect to transfer of the sulfuryl group
transfer. Either the spatial arrangement of the methyl
group on the hydroxymethyl substituent may block the
approach of the sulfuryl group to the benzylic oxygen, or
the hydroxyl group is too distant from the sulfuryl group
of PAPS for transfer to occur. For sulfuryl group transfer
to occur, conformational rotation would be required,
wherein one of the substituents at the benzylic position
would have to pass the peri-hydrogen to fulfill the
conformational requirements for reaction. Such a passage
F igu r e 4. Conformational energies as a function of torsion
angle ω. A torsion angle of 0° is defined as the conformation
where the oxygen, benzylic carbon, and aromatic ring system
all lie in the same plane with the oxygen at a maximum distance
from the peri-hydrogen. Energies for the various conformations
were calculated as described in Materials and Methods. Data
are represented as follows: (s) (S)-(-)-1-(1-naphthyl)ethanol,
(‚‚‚) (S)-(-)-1-(9-phenanthryl)ethanol, (- -) (S)-(-)-1-(1-pyre-
nyl)ethanol, and (-‚-) (S)-(-)-1-(2-naphthyl)ethanol. Represen-
tative conformations are illustrated (left to right) for ω values
of 60, 120, (180, and -30°. In these illustrations, the broken
line indicates the plane of the aromatic ring system, with the
longer portion of the line representing the portion of the
molecule containing the peri-hydrogen.
thyl)ethanol. Indeed, the energy differences are very large
for 1-(1-naphthyl)-, 1-(1-pyrenyl)-, and 1-(9-phenanthryl)-
ethanol, where the spatial rearrangements of the benzylic
hydroxyl and methyl group at the 1-position (9-position
on phenanthrene ring) are obstructed by the adjacent
peri-hydrogen. This strongly suggests that the differences
in the interaction of enantiomers of these molecules at
the active site of STa are conformational in origin.
Discu ssion
Recent reports on the activation of PAHs bearing a
benzylic hydroxyl functional group have indicated that
hydroxysteroid (alcohol) sulfotransferases play an im-
portant role in the metabolic activation of these mol-
ecules. In addition, since the formation of these PAHs
bearing benzylic alcohol groups is often mediated ste-
reoselectively by cytochrome P450s and epoxide hydro-
lases (36), the stereoselective formation of sulfate con-
jugates is likely to be an important factor in the sulfation-
mediated DNA binding of chiral hydroxyalkyl polycyclic
aromatic hydrocarbons. This was further supported by
Glatt and his colleagues in studies with the human
hydroxysteroid sulfotransferase- and rat STa-mediated
metabolic activation of the stereoisomers of 1-(1-pyrenyl)-
ethanol in Salmonella typhimurium strains (37). Their
results with STa heterologously expressed in a strain of
S. typhimurium showed that (R)-(+)-1-(1-pyrenyl)ethanol
caused a mutagenic response 2-fold greater than that
caused by the (S)-(-)-enantiomer. One of the possible
reasons for such stereochemical differences is an enan-
tiomeric selectivity by the enzyme(s) catalyzing the
sulfation of 1-arylethanols. Indeed, enantioselectivity
with 1-(1-pyrenyl)ethanol has been recently observed for
heterologously expressed human hydroxysteroid sulfo-
transferase and human estrogen sulfotransferase (38).
Our investigations on the stereochemistry of sulfation
of 1-arylethanols have centered on STa, the major hepatic

284 Chem. Res. Toxicol., Vol. 12, No. 3, 1999
Banoglu and Duffel
steroid sulfotransferases catalysing covalent binding of carcino-
genic polycyclic arylmethanols to DNA. Chem.-Biol. Interact. 92,
87-105.
would involve a prohibitively severe energy barrier
(Figure 4).
In addition to the orientation of the benzylic hydroxyl
and methyl groups, the hydrophobic characteristics of
these benzylic alcohols contribute to their ability to
interact with the active site of the STa. Partition coef-
ficients (log P) were calculated for 1-(1-naphthyl)ethanol
(log P ) 2.6), 1-(9-phenanthryl)ethanol (log P ) 3.84),
and 1-(1-pyrenyl)ethanol (log P ) 4.33). Values of appar-
ent Km [800 ( 100 µM for 1-(1-naphthyl)ethanol, 90 (
20 µM for 1-(9-phenanthryl)ethanol, and 7 ( 0.6 M for
1-(1-pyrenyl)ethanol] decreased in relation to increasing
partition coefficient values. Although these data were
determined with only a few compounds, they were
consistent with more extensive previous studies (23) on
the role of hydrophobic interactions in the specificity of
STa.
(9) Ogura, K., Sohtome, T., Sugiyama, A., Okuda, H., Hiratsuka, A.,
and Watabe, T. (1990) Rat liver cytosolic hydroxysteroid sul-
fotransferase (sulfotransferase a) catalyzing the formation of
reactive sulfate esters from carcinogenic polycyclic hydroxy-
methylarenes. Mol. Pharmacol. 37, 848-854.
(10) Miller, J . A., and Surh, Y.-J . (1994) Sulfonation in chemical
carcinogenesis. In Handbook of Experimental Pharmacology
(Kauffman, F. C., Ed.) Vol. 112, pp 429-457, Springer-Verlag,
Berlin.
(11) Miller, J . A. (1994) Sulfonation in chemical carcinogenesis-history
and present status. Chem. Biol. Interact. 92, 329-341.
(12) Chou, H.-C., Ozawa, S., Fu, P. P., Lang, N. P., and Kadlubar, F.
F. (1998) Metabolic activation of methyl-hydroxylated derivatives
of 7,12-dimethylbenz[a]anthracene by human liver dehydroepi-
androsterone-steroid sulfotransferase. Carcinogenesis 19, 1071-
1076.
(13) Watabe, T., Ishizuka, T., Isobe, M., and Ozawa, N. (1982) A
7-hydroxymethyl sulfate ester as an active metabolite of 7,12-
dimethylbenz[a]anthracene. Science 215, 403-405.
In conclusion, conformational restrictions due to peri-
substituent interactions combine with hydrophobic in-
teractions between the enzyme and substrate to deter-
mine the specificity of hydroxysteroid sulfotransferase
STa for these peri-substituted arylethanols. These results
will furnish a foundation for more extensive investiga-
tions on the molecular interactions that are required for
sulfotransferase-mediated activation of chemical carcino-
gens and other xenobiotics that possess chiral benzylic
alcohol functional groups. Such studies will undoubtedly
be aided by the recently published crystallographic
studies on the structure (45) and mechanism (46) of the
mouse estrogen sulfotransferase. However, although one
expects a relatively high correlation between the gross
three-dimensional folding of an HST and an estrogen
sulfotransferase, structure-activity relationships such as
those defined here for 1-arylethanols provide an essential
component in the development and refinement of homol-
ogy models for the active sites of HSTs that are useful
in predicting the specificity of the enzymes for substrates
and inhibitors.
(14) Watabe, T., Fujieda, T., Hiratsuka, A., Ishizuka, T., Hakamata,
Y., and Ogura, K. (1985) The carcinogen, 7-hydroxymethyl-12-
methylbanz[a]anthracene, is activated and covalently binds to
DNA via a sulfate ester. Biochem. Pharmacol. 34, 3002-3005.
(15) Glatt, H., Christoph, S., Czich, A., Pauly, K., Vierzok, A., Seidel,
A., Coughtrie, M. W. H., Doehmer, J ., Falany, C. N., Philips, D.
H., Yamazoe, Y., and Bartsch, I. (1997) Rat and human sulfo-
transferases expressed in Ames Salmonella typhimurium strains
and Chinese hamster V79 cells for the activation of mutagens.
In Control Mechanisms of Carcinogenesis (Hengstler, J . G., and
Oesch, F., Eds.) pp 98-115, Druckerei Thieme, Meissen.
(16) Okuda, H., Nojima, H., Watanabe, N., and Watabe, T. (1989)
Sulphotransferase-mediated activation of the carcinogen 5-hy-
droxymethyl-chrysene. Biochem. Pharmacol. 38, 3003-3009.
(17) Surh, Y.-J ., Liem, A., Miller, E. C., and Miller, J . A. (1989)
Metabolic activation of the carcinogen 6-hydroxymethylbenzo[a]-
pyrene: formation of an electrophilic sulfuric acid ester and
benzylic DNA adducts in rat liver in vivo and reactions in vitro.
Carcinogenesis 10, 1519-1528.
(18) Surh, Y.-J ., Blomquist, J . C., Liem, A., and Miller, J . A. (1990)
Metabolic activation of 9-hydroxymethyl-10-methylbenzanthracene
and 1-hydroxymethylpyrene to electrophilic, mutagenic, and
tumorigenic sulfuric acid esters by rat hepatic sulfotransferase
activity. Carcinogenesis 11, 1451-1460.
(19) Surh, Y.-J ., Lai, C.-C., Miller, J . A., and Miller, E. C. (1987)
Hepatic DNA and RNA adduct formation from 7-hydroxymethyl-
12-methylbenz[a]anthracene and its electrophilic sulfuric acid
ester metabolite in preweanling rats and mice. Biochem. Biophys.
Res. Commun. 144, 576-582.
(20) Surh, Y.-J ., Kwon, H., and Tannenbaum, S. R. (1993) Sulfotrans-
ferase-mediated activation of 4-hydroxy- and 3,4-dihydroxy-3,4-
dihydrocyclopenta[c,d]pyrene, major metabolites of cyclopenta-
[c,d]pyrene. Cancer Res. 53, 1017-1022.
(21) Glatt, H., Pauly, K., Frank, H., Seidel, A., Oesch, F., Harvey, R.
G., and Werle-Schneider, G. (1994) Substance-dependent sex
differences in the activation of benzylic alcohols to mutagens by
hepatic sulfotransferases of the rat. Carcinogenesis 15, 2605-
2611.
(22) Chen, G., Banoglu, E., and Duffel, M. W. (1996) Influence of
substrate structure on the catalytic efficiency of hydroxysteroid
sulfotransferase STa in the sulfation of alcohols. Chem. Res.
Toxicol. 9, 67-74.
(23) Banoglu, E., and Duffel, M. W. (1997) Studies on the interactions
of chiral secondary alcohols with hydroxysteroid sulfotransferase
STa. Drug Metab. Dispos. 25, 1304-1310.
Ack n ow led gm en t. We gratefully acknowledge the
assistance in crystallographic analysis provided by Dr.
Dale C. Swenson of The University of Iowa X-ray
Structure Facility. This investigation was supported by
U.S. Public Health Service Grant CA38683 awarded by
the National Cancer Institute, Department of Health and
Human Services.
Refer en ces
(1) Strott, C. A. (1996) Steroid sulfotransferases. Endocr. Rev. 17,
670-697.
(2) Duffel, M. W. (1997) Sulfotransferases. In Comprehensive Toxicol-
ogy Volume 3, Biotransformation (Guengerich, F. P., Ed.) pp 365-
383, Elsevier, Oxford, U.K.
(3) Mulder, G. J ., and J akoby, W. B. (1990) Sulfation. In Conjugation
Reactions in Drug Metabolism (Mulder, G. J ., Ed.) pp 107-161,
Taylor & Francis, New York.
(24) Sekura, R. D. (1981) Adenosine 3′-phosphate 5′-phosphosulfate.
Methods Enzymol. 77, 413-415.
(4) Luu-Thee, V., Bernier, F., and Dufort, I. (1996) Steroid sulfotrans-
ferases. J . Endocrinol. 150, S87-S97.
(5) Yamazoe, Y., Nagata, K., Ozawa, S., and Kato, R. (1994) Struc-
tural similarity and diversity of sulfotransferases. Chem.-Biol.
Interact. 92, 107-117.
(6) Rikke, B. A., and Roy, A. K. (1996) Structural relationships among
members of the mammalian sulfotransferase gene family. Bio-
chim. Biophys. Acta 1307, 331-338.
(7) Weinshilboum, R. M., Otterness, D. M., Aksoy, I. A., Wood, T.
C., Her, C., and Raftogianis, R. B. (1997) Sulfation and sulfo-
transferases 1: Sulfotransferase molecular biology: cDNAs and
genes. FASEB J . 11, 3-14.
(25) Urbanski, T., and Wolff, M. (1965) 1-Acenaphthenone and its
derivatives. I. A selective oxidation of acenaphthene and its nitro
derivatives. Rocz. Chem. 39, 1447-1453.
(26) Kato, K., Katayama, M., Fujii, S., and Kimoto, H. (1995) Optical
resolution of 1-arylethanols with a condensed aromatic ring by
lipase from Pseudomonas aeruginosa. Biosci., Biotechnol., Bio-
chem. 59, 2178-2180.
(27) Chandrasekharan, J ., Ramachandran, P. V., and Brown, H. C.
(1985) Diisopinocampheylchloroborane, a remarkably efficient
chiral reducing agent for aromatic prochiral ketones. J . Org.
Chem. 50, 5446-5448.
(28) Hu, Y., Ziffer, H., and Silverton, J . V. (1989) Preparation and
absolute stereochemistry of (-)-acenaphthenol. Can. J . Chem. 67,
60-62.
(8) Watabe, T., Ogura, K., Satsukawa, M., Okuda, H., and Hiratsuka,
A. (1994) Molecular cloning and functions of rat liver hydroxy-

Stereospecificity of Sulfotransferase STa
Chem. Res. Toxicol., Vol. 12, No. 3, 1999 285
(29) States, D. J ., Haberkorn, R. A., and Ruben, D. J . (1982) Hyper-
complex phase cycling. J . Magn. Reson. 48, 286-292.
(30) Kessler, H. (1987) Separation of cross-relaxation and J cross-peaks
in 2D rotating-frame NMR spectroscopy. J . Am. Chem. Soc. 109,
607-609.
(31) Bensadoun, A., and Weinstein, D. (1976) Assay of proteins in the
presence of interfering materials. Anal. Biochem. 70, 241-250.
(32) Duffel, M. W., Binder, T. P., and Rao, S. I. (1989) Assay of purified
aryl sulfotransferase suitable for reactions yielding unstable
sulfuric acid esters. Anal. Biochem. 183, 320-324.
tiomers mediated by enantioselective sulfotransferases. Biochem.
Biophys. Res. Commun. 247, 181-185.
(39) Balasubramaniyan, V. (1966) peri Interaction in naphthalene
derivatives. Chem. Rev. 66, 567-633.
(40) Bentley, M. D., and Dewar, M. J . S. (1970) Proton nuclear
magnetic resonance spectra of arylmethyl systems. J . Org. Chem.
35, 2707-2710.
(41) Chen, P. H., and Kleinfelter, D. C. (1973) Peri effects in the mass
spectra of some 8-substituted 1-naphthoic acids and 1-naphthyl-
carbinols. J . Org. Chem. 38, 3015-3019.
(33) Perella, F. W. (1988) EZ-FIT: A practical curve-fitting microcom-
puter program for the analysis of enzyme kinetic data on IBM-
PC compatible computers. Anal. Biochem. 174, 437-447.
(34) Ogura, K., Kajita, J ., Narihata, H., Watabe, T., Ozawa, S., Nagata,
K., Yamazoe, Y., and Kato, R. (1990) cDNA cloning of the
hydroxysteroid sulfotransferase STa sharing a strong homology
in amino acid sequence with the senescence marker protein
SMP-2 in rat livers. Biochem. Biophys. Res. Commun. 166, 1494-
1500.
(35) Segel, I. H. (1975) Competitive inhibition and total velocity with
mixed alternative substrates. In Enzyme Kinetics, pp 113-118,
Wiley-Interscience, New York.
(36) Yang, S. K. (1988) Stereoselectivity of cytochrome P-450 isozymes
and epoxide hydrolase in the metabolism of polycyclic aromatic
hydrocarbons. Biochem. Pharmacol. 37, 61-70.
(42) Kleinfelter, D. C., and Chen, P. H. (1969) Proximity effects in
peri-substituted naphthalenes. I. Some 8-substituted 1-hydroxy-
methyl- and 1-chloromethylnaphthalenes. J . Org. Chem. 34,
1741-1745.
(43) Ouellette, R. J ., Marks, D. L., and Miller, D. (1967) Conforma-
tional analysis. IX. Conformational and substituent dependence
of the hydroxyl proton magnetic resonance of arylcarbinols. J . Am.
Chem. Soc. 89, 913-917.
(44) Ouellette, R. J ., and VanLeuwen, B. G. (1969) Conformational
analysis-XIII polycyclic arylmethanols. Tetrahedron 25, 819-825.
(45) Kakuta, Y., Pedersen, L. G., Carter, C. W., Negishi, M., and
Pedersen, L. C. (1997) Crystal structure of estrogen sulfotrans-
ferase. Nat. Struct. Biol. 4, 904-908.
(46) Kakuta, Y., Petrotchenko, E. V., Pedersen, L. C., and Negishi,
M. (1998) The sulfuryl transfer mechanism. Crystal structure of
a vanadate complex of estrogen sulfotransferase and mutational
analysis. J . Biol. Chem. 273, 27325-27330.
(37) Glatt, H. R. (1997) Bioactivation of mutagens via sulfation.
FASEB J . 11, 314-321.
(38) Landsiedel, R., Pabel, U., Engst, W., Ploschke, J ., Seidel, A., and
Glatt, H. (1998) Chiral inversion of 1-hydroxyethylpyrene enan-
TX980219F