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Abstract |
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 Top Abstract Materials and MethodsResults and DiscussionReferences |
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; Jakoby and Ziegler,
1990; Duffel, 1997;
Falany, 1997;
Weinshilboum et al., 1997;
Nagata and Yamazoe, 2000;
Duffel et al., 2001;
Glatt et al., 2001). The
reactions involve the transfer of a sulfuryl group from
3'-phosphoadenosine 5'-phosphosulfate
(PAPS1) to a hydroxyl
or amino functional group of an acceptor molecule, and the products are
adenosine 3', 5'-diphosphate (PAP), and a sulfuric acid ester,
respectively.
Hydroxysteroid (alcohol) sulfotransferases, enzymes constituting a
subfamily of the mammalian cytosolic sulfotransferases, catalyze the sulfation
of a wide variety of hydroxylated endobiotic and xenobiotic compounds
(Lyon et al., 1981;
Ogura et al., 1990;
Falany et al., 1994;
Glatt, 1997;
Kudlacek et al., 1997;
Shibutani et al., 1998;
Lewis et al., 2000). In
addition to hydroxysteroids, the xenobiotic substrates of hydroxysteroid
sulfotransferases include many benzylic alcohols derived from
alkyl-substituted polycyclic aromatic hydrocarbons, environmental contaminants
known to induce mutations and tumors in both experimental animals and humans
(Watabe et al., 1982;
Surh et al., 1990;
Miller and Surh, 1994;
Yamazoe et al., 1999).
Hydroxysteroid (alcohol) sulfotransferase was first purified to homogeneity
from rat liver by Jakoby and coworkers
(Lyon and Jakoby, 1980). To
date, the cDNAs of hydroxysteroid sulfotransferases have been identified from
both human and rodent species (Ogura et
al., 1989; Kong et al.,
1992; Comer et al.,
1993; Otterness and
Weinshilboum, 1994; Sakakibara
et al., 1998; Chang et al.,
2001), and the crystal structure of a human hydroxysteroid
sulfotransferase has been determined
(Pedersen et al., 2000;
Rehse et al., 2002).
The influence of structural characteristics of various substrates on the
catalytic efficiency of a hydroxysteroid sulfotransferase from rat liver has
been studied previously (Chen et al.,
1996; Banoglu and Duffel,
1997,
1999;
Sheng and Duffel, 2001). One
of these important characteristics is the stereochemistry of the molecule that
serves as the sulfuryl acceptor. This substrate-stereoselectivity has been
found in the sulfation of several model chiral secondary alcohols catalyzed by
the rat hepatic hydroxysteroid sulfotransferase
(Chen et al., 1996; Banoglu and
Duffel, 1997,
1999). We now report on the
stereoselectivity of the human hydroxysteroid sulfotransferase, ST2A3, using
the enantiomers of 1- and 2-naphthyl-1-ethanols
(Fig. 1) as model
substrates.
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Materials and Methods |
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TopAbstractMaterials and MethodsResults and DiscussionReferences |
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Prokaryotic Expression and Purification of Human Hydroxysteroid
Sulfotransferase. For overexpression and purification, the human
hydroxysteroid sulfotransferase cDNA coding region was transferred from a cDNA
clone in a pKK233–2 vector (Comer et
al., 1993) (EMBL accession number: XM_049895, kindly provided by
Dr. Charles Falany at the University of Alabama at Birmingham) into a pET-3c
(with a T7 promotor; Novagen, Madison, WI) vector between the NdeI
restriction site and the HindIII site. The newly constructed
expression vector was transformed into Escherichia coli BL21 (DE3)
cells (Novagen). The bacterial cell cultures were grown from single colonies
in 20 ml of Luria broth containing 50 µg/ml ampicillin. After incubation
overnight at 37°C, the culture was used to inoculate 500 ml of the Luria
broth medium with 1 mM isopropyl-1-thio-D-galactopyranoside. The
second stage culture was incubated on a reciprocating shaker (250 rpm)
overnight at 30°C. The cells were harvested by centrifugation at
10,000g for 30 min. The cell pellet, about 3.8 g (wet weight) was
suspended in 25 ml of ice-cold buffer A (25 mM Tris-HCl buffer, pH 7.4,
containing 0.25 M sucrose, 10% (v/v) glycerol, 1 mM phenylmethylsulfonyl
fluoride, 1 µM pepstatin A, and 1 mM dithiothreitol). Cells in this
suspension were then disrupted at 4°C with a Sonifier model 350 (Bronson
Sonic Power Company, Danbury, CT). The sonicator was programmed to provide
three sonication periods of 90 s each (using 2-s cycle pulses at 50% power
output), with 30 s "off-time" between sonication periods. The
supernatant fluid was collected after centrifugation at 24,000g for
30 min. Solid PEG8000 was then slowly added with stirring to the supernatant
until a final concentration of 5% (w/v) was reached. The solution was stirred
at 4°C for 30 min before centrifugation at 24,000g for 30 min.
The pellet was discarded, and more PEG8000 was added to the supernatant
fraction to reach a concentration of 40% (w/v). After stirring for 30 min at
4°C, the mixture was subjected to centrifugation at 24,000g for
30 min. The resulting pellet was dissolved in 10 ml of buffer A. This
extraction and precipitation procedure was repeated two more times, and the
final volume of the precipitated protein solution was 30 ml.
Chromatography on PAP-agarose was used to purify the recombinant ST2A3. The
resuspended precipitate of 40% PEG8000 was charged onto a column of
PAP-agarose (5 ml) that had been previously equilibrated with buffer A. The
column was washed with 250 ml of buffer A, and the recombinant protein was
eluted from the column with 30 ml of buffer A containing 250 µM PAP. The
effluent was diluted with 10 volumes of buffer A followed by ultrafiltration
(PM-10 membrane; Millipore Corporation, Bedford, MA) for three times to remove
the excess PAP remaining in the enzyme solution. The final concentration of
PAP in the enzyme solution was 0.3 µM. The enzyme solution was then
concentrated by ultrafiltration (PM-10 membrane), and the protein
concentration was determined using a modified Lowry procedure
(Bensadoun and Weinstein,
1976), with bovine serum albumin as standard. The resulting ST2A3
was highly purified (apparent homogeneity by SDS-polyacrylamide gel
electrophoresis with Coomassie blue staining). It shared similar mobility on
SDS-polyacrylamide gel electrophoresis with that reported for the native
hydroxysteroid sulfotransferase purified from human liver tissue
(Falany et al., 1989).
Specific activity of the purified ST2A3 (assay at pH 5.5) was 106 nmoles DHEA
sulfate formed per minute per milligram of enzyme.
Assays for Catalytic Activity of ST2A3. During expression and
purification, the enzymatic activity was monitored with DHEA as substrate
using a methylene blue assay procedure described previously
(Nose and Lipmann, 1958;
Sheng et al., 2001). In
addition to the ST2A3, assay mixtures contained 0.25 M sodium acetate (pH
5.5), 0.050 mM DHEA, 7.5 mM 2-mercaptoethanol, and 0.20 mM PAPS. Reactions
were carried out at 37°C for 30 min.
The kinetic constants of the recombinant protein in the sulfation of the
stereoisomers of 1- and 2-naphthyl-1-ethanol were determined at pH 7.0 using a
published HPLC method for determination of substrate-dependent formation of
PAP in the reaction (Duffel et al.,
1989; Sheng et al.,
2001). This procedure has proven to be especially useful for
reactions resulting in the formation of benzylic sulfates such as 1- and
2-naphthyl-1-ethanol where stability of the product often limits accurate
determination of the rate of sulfation. Reaction mixtures of 40 µl of total
volume contained 0.25 M potassium phosphate buffer (pH 7.0), 7.5 mM
2-mercaptoethanol, 0.20 mM PAPS, and various concentrations of the substrates
dissolved in acetone (the final concentration of acetone in the assay was 2.5%
v/v). The reaction mixture with the same amount of acetone but no substrate
added was used as control. Both control and experimental reactions were
initiated by addition of enzyme, incubated at 37°C for 30 min, and
terminated by addition of 40 µl of methanol. The substrate-dependent
formation of PAP in the reaction was determined by HPLC. Analyses were carried
out on an Econosphere C18 column (300-Å, 5 µ, 4.6 mm
x 250 mm; Alltech, Deerfield, IL), with a mobile phase of water/methanol
(88:12, v/v) containing 65 mM KH2PO4 (pH 5.45), 1.0 mM
1-octylamine, and 65 mM NH4Cl. A flow rate of 2 ml/min and
detection at 254 nm was used for all determinations of PAP. The concentration
of PAP formed in a reaction mixture was determined from the HPLC peak area
using a linear standard curve relating peak area to the concentration of PAP.
The limit of detection of substrate-dependent PAP formation was 0.1
nmol/min/mg. At least six concentrations of each substrate were used and these
included concentrations both greater than and less than the apparent
Km. Apparent Km and
Vmax values were obtained by fitting the initial rate data
to the folowing equation: 
= Vmax
* [S]/(Km + [S]). The
apparent Km and Vmax values are
presented as the mean ± the standard error.
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Results and Discussion |
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TopAbstractMaterials and MethodsResults and DiscussionReferences |
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; Sheng and Duffel,
2001). We have now explored stereospecificity of the homologous
human enzyme, ST2A3, by evaluating the R-(+)-and
S-(-)-enantiomers of both 1-naphthyl-1-ethanol and
2-naphthyl-1-ethanol as model substrates. Stereospecificity was seen in the
sulfation of enantiomers of 1-naphthyl-1-ethanol catalyzed by ST2A3, wherein
the R-(+)-isomer was a substrate, but the S-(-)-isomer was
not (Table 1). Although the
S-(-)-enantiomer of 1-naphthyl-1-ethanol was not a substrate, it was
still able to interact with the ST2A3 enzyme and inhibit catalytic activity
toward DHEA. The specific activity of ST2A3 in the sulfation of DHEA was
significantly decreased upon addition of 500 µM
S-(-)-1-naphthyl-1-ethanol (Fig.
2). In contrast, stereoselectivity was not seen in the sulfation
of enantiomers of 2-naphthyl-1-ethanol. Thus, the initial velocities of both
R-(+)- and S-(-)-isomers were similar, and the ratio of the
kcat/Km values were only slightly
different (Table 1).
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These results are analogous to those seen with the homologous enzyme from
rat liver (Banoglu and Duffel,
1999), in which steric interactions between the ethanol
functionality and a hydrogen atom at the peri-position (carbon 8) on
the naphthyl ring system (Fig.
1) play an important role in determining substrate
stereoselectivity for 1-naphthyl-1-ethanol. It has long been recognized that
there are significant steric interactions between substituents on the benzylic
carbon of 1-alkylsubstituted naphthalenes and the hydrogen in the C-8 position
of the naphthalene ring (Balasubramaniyan,
1966). Moreover, other alkyl-substituted arenes also exhibit this
effect (Bentley and Dewar,
1970).
Although it is apparent that the interaction of
R-(+)-1-naphthyl-1-ethanol is catalytically productive, the exact
nature of the interaction between the S-(-)-1-naphthyl-1-ethanol and
the ST2A3 is not yet clear. Nonetheless, preliminary molecular docking studies
(not shown) on the naphthyl-1-ethanols modeled in the sulfuryl acceptor site
of the crystal structure coordinates of ST2A3 do suggest a potential
explanation for further analysis. That is, the naphthyl ring of
S-(-)-1-naphthyl-1-ethanol may bind to the sulfuryl acceptor site in
a position similar to that occurring in the binding of
R-(+)-1-naphthyl-1-ethanol and both enantiomers of
2-napthyl-1-ethanol. However, in such a case, the peri-interactions
with S-(-)-1-naphthyl-1-ethanol would prevent conformational changes
that would be necessary for proper alignment of the benzylic hydroxyl for
catalysis to occur. As noted in previous studies
(Kakuta et al., 1998), such
alignment between the oxygen of an acceptor molecule and the sulfuryl group,
as well as the assistance of a conserved histidine residue in proton
abstraction, is essential for catalysis. With the 2-naphthyl-1-ethanols, there
are no such peri-interactions
(Fig. 1), and conformational
rotation is allowed for the hydroxyl group to be in a catalytically favorable
position for accepting the sulfuryl group from PAPS. Thus, the
stereospecificity of ST2A3 for the R-(+)-isomer of
1-naphthyl-1-ethanol as substrate is consistent with the suggestion that a
major determinant of binding orientation for these naphthyl-1-ethanols at the
active site of the enzyme is the position of the naphthyl ring system.
Although the use of rodent enzymes as models for catalytic function of
human enzymes is usually subject to significant interspecies differences, the
observed stereochemical properties of both the rat and human sulfotransferases
with these model naphthylethanols suggest that previous results on the
specificity of the rat hydroxysteroid sulfotransferase for chiral benzylic
alcohols (Chen et al., 1996;
Banoglu and Duffel, 1997,
1999) may have direct relevance
to the human enzyme. This may be particularly applicable to those hydroxyalkyl
polycyclic aromatics where peri-interactions constrain conformational
rotation. Future studies will focus on better definition of the full extent of
these parallels in stereospecificity as well as the identification of specific
key amino acid residues at the enzyme active sites that contribute to the
stereoselectivity of both the rat and human hydroxysteroid sulfotransferases
with chiral alcohols that exhibit varying degrees of conformational
restriction.
Jonathan J. Sheng
Michael W. Duffel
Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa, Iowa City, Iowa
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Acknowledgments |
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Footnotes |
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1 Abbreviations used are: PAPS, 3'-phosphoadenosine
5'-phosphosulfate; PAP, adenosine 3',5'-diphosphate; DHEA,
dehydroepiandrosterone; PEG8000, polyethylene glycol 8000; HPLC, high
performance liquid chromotography; EMBL, European Molecular Biology
Laboratory; ST2A3, hDHEA-ST, and SULT2A1, human hydroxysteroid
sulfotransferase (EMBL accession number XM_049895; PDB code 1EFH
[PDB]
); ST2A2, rat
hydroxysteroid sulfotransferase (EMBL accession number M33329
[GenBank]
). 
Address correspondence to: Michael W. Duffel, Division of Medicinal and Natural Products Chemistry, College of Pharmacy, The University of Iowa, Iowa City, IA 52242. E-mail: michael-duffel{at}uiowa.edu
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References |
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TopAbstractMaterials and MethodsResults and DiscussionReferences |
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