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Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa, Iowa City, Iowa
(Received October 10, 2003; Accepted January 16, 2004)
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Abstract |
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; Weinshilboum and Otterness, 1994; Falany, 1997; Duffel et al., 2001). Additionally, these enzymes catalyze the sulfation of endogenous hormones, such as thyroid hormone T3 (3,5,3'-triiodothyronine) and related metabolites (Sekura et al., 1981), as well as neurotransmitters such as epinephrine and dopamine (Jakoby et al., 1980). In all cases, the aryl sulfotransferases catalyze the transfer of a sulfuryl group from the donor substrate 3'-phosphoadenosine 5'-phosphosulfate (PAPS1) to an acceptor substrate. Although sulfotransferase-catalyzed reactions with most endogenous and xenobiotic substrates yield sulfuric acid esters that are less biologically active and more readily excreted, there are significant exceptions with important implications for carcinogenesis and other toxic responses to xenobiotics (Miller, 1994; Duffel, 1997; Glatt et al., 2001).
The primary sequences of many aryl sulfotransferases have now been deduced from cDNA analysis, and it has therefore been possible to develop classification schemes for these enzymes in relation to other cytosolic sulfotransferases (Weinshilboum et al., 1997; Nagata and Yamazoe, 2000; Honma et al., 2001). The human cytosolic sulfotransferases have been classified according to families and subfamilies preceded by the prefix "SULT" (Weinshilboum et al., 1997), and a nomenclature for isoforms of these enzymes in all mammalian species has been proposed wherein the prefix "ST" precedes family and subfamily designation (Nagata and Yamazoe, 2000). Crystallographic analyses of several aryl sulfotransferases have greatly contributed to our understanding of the structure and function of these enzymes (Kakuta et al., 1997, 1998; Bidwell et al., 1999; Dajani et al., 1999; Negishi et al., 2001; Yoshinari et al., 2001; Pedersen et al., 2002; Gamage et al., 2003). In addition to important advances in structural details of the mechanism of sulfuryl transfer, it is also clear that the three-dimensional structures of sulfotransferases retain a high degree of homology, even as the primary sequences exhibit decreasing identity (Negishi et al., 2001). Thus, it becomes increasingly important to discern the roles of individual amino acid residues that determine molecular recognition of substrates and inhibitors of these enzymes.
The major form of cytosolic aryl sulfotransferase present in rat liver is AST IV (also known as tyrosine-ester sulfotransferase or ST1A1) (Jakoby et al., 1980). AST IV has a remarkably broad specificity for sulfuryl acceptors that possess a variety of oxygen-containing functional groups. Nevertheless, within this large capacity for various types of sulfuryl acceptors, there is a distinct stereospecificity for the sulfation of many chiral benzylic alcohols (Rao and Duffel, 1991; Duffel, 1994), particularly those with sterically bulky substituents on the chiral carbon. In addition to its importance in a better understanding of the role of stereochemistry in pathways of drug and xenobiotic metabolism, the use of stereospecific substrates and inhibitors also provides an important tool for investigation of the catalytic specificity and pharmacological function of sulfotransferases. Investigations on the stereochemical properties of AST IV are particularly important in light of the extensive use of the rat for model studies in drug metabolism and carcinogenesis, as well as the high structural and functional homology of this isoform with human aryl sulfotransferases.
In the present studies, enantiomers of 1,2,3,4-tetrahydro-1-naphthol (structures shown in Fig. 1) are used as chiral probes to examine the potential role(s) of three conserved aromatic residues in enzyme-substrate interactions at the sulfuryl acceptor binding site of AST IV. Furthermore, results are analyzed within the context of molecular modeling studies using a homology model of AST IV.
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Materials and Methods |
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Site-Directed Mutagenesis. The bacterial expression vector pET-3c containing AST IV cDNA obtained from previous studies (Chen et al., 1992) was used as a template to generate mutant constructs. The mutant oligonucleotide primers (5'-CCGGGTACCCGCCCTTGAGTTCA-3' for F77A, 5'-TCCTATTATAACGCCTACAACATGGC-3' for F138A, and 5'-CATGACTAACGCCACAACCATCC-3' for Y236A) were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA). The mutations were performed with a Transformer site-directed mutagenesis kit (BD Biosciences Clontech, Palo Alto, CA). In each case, the oligonucleotide primer was annealed to one strand of the wild-type template DNA along with another oligonucleotide (5'-TTTGCTCACATCGTCTTTCCTGCG-3') to generate a mutant strand. Subsequently, synthesis and ligation were performed to link them. The DNA was then transformed into a repair minus strain of Escherichia coli (BMH 71-18 mutS) (BD Biosciences Clontech). AflIII digestion was carried out to selectively linearize the parental vector before the transformation step. A second round of AflIII selection and transformation into JM109 E. coli cells (Novagen, Madison, WI) was then performed. Bacterial colonies containing the desired mutation were identified by AflIII restriction mapping, and sequence analyses.
Prokaryotic Expression of AST IV. Either the wild-type or mutant expression vector, as appropriate, was transformed into E. coli BL21 (DE3) cells. The bacterial cell cultures were grown from single colonies in 5 ml of LB containing 50 µg/ml ampicillin. After incubation overnight at 30°C, the culture was used to inoculate 300 ml of the LB medium containing 1 mM isopropyl-1-thio-D-galactopyranoside. This second-stage culture was incubated on a reciprocating shaker (250 rpm) overnight at 30°C. When the culture reached an absorbance at 600 nm of about 6.0, the cells were harvested by centrifugation at 10,000g for 30 min. The cell pellets were stored at -70°C until used.
Preparation of Crude Cell Extract. The cells from 250 ml of bacterial culture were harvested by centrifugation as noted above, and the cell pellet was suspended in 15 ml of ice-cold buffer A [25 mM Tris HCl, 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 were then disrupted at 4°C with a Sonifier cell disrupter model 350 (Branson Sonic Power Company, Danbury, CT). The sonicator was programmed to provide three periods of 90 s (2-s cycle pulses at 50% power output during each 90-s period) with 30 s of "off-time" between the periods. The supernatant fluid was collected after centrifugation at 24,000g for 30 min.
Affinity Purification of the Recombinant AST IV. Chromatography on PAP-agarose (Sigma-Aldrich) was used to purify the wild-type and mutant AST IV enzymes. The cell extract was charged onto a column of PAP-agarose (5 ml) that had been previously equilibrated with buffer A. The direction of flow for the column was reversed after the column was washed with 100 ml of buffer A. The column was then washed with an additional 30 ml of buffer A, and the recombinant protein was eluted from the column with 15 ml of buffer A containing 500 µM PAP. The effluent was then concentrated to approximately 2 ml by ultrafiltration (PM-10 membrane, Millipore Corporation, Billerica, MA). Excess PAP remaining in the enzyme solution was extracted by chromatography on a disposable PD-10 column (Amersham Biosciences, Inc., Piscataway, NJ) that was pre-equilibrated with buffer A. The PD-10 column was eluted with 10 ml of buffer A. The fractions containing the AST IV enzymes were collected, concentrated to about 1 ml, and stored at - 70°C. The protein concentration was determined using a modified Lowry procedure (Bensadoun and Weinstein, 1976), with bovine serum albumin as standard.
SDS-PAGE and Western Blot Analysis. SDS-PAGE was performed in 12% gels with a Bio-Rad Mini-Protein II apparatus (Bio-Rad, Hercules, CA) as described by Laemmli (1970). Before SDS-PAGE, the purified proteins were dialyzed to remove the excess sucrose and glycerol in the enzyme solution using a YM-10 ultrafiltration membrane (Millipore) and a pH 7.4 buffer containing 140 mM sodium chloride, 2.7 mM potassium chloride, 10 mM sodium phosphate, and 1.8 mM potassium phosphate. After SDS-PAGE, the protein bands were either stained by Coomassie Blue R-250 or analyzed by Western blotting as described previously (Chen et al., 1995).
Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry. For protein analyses, 2 µg of the wild-type or mutant AST IV proteins was purified by polyacrylamide gel and digested with 7 ng of trypsin (Promega, Madison, WI) in 10 mM NH4HCO3, pH 8.0 for 18 to 20 h at 37°C. Samples were diluted 1:1 in 50% acetonitrile/0.1% (v/v) trifluoroacetic acid and air-dehydrated with 
-cyano-4-hydroxycinnaminic acid (matrix) before analysis. Mass determination was performed with a Bruker Biflex III MALDITOF mass spectrometer (Bruker Daltonics Inc., Billerica, MA). All spectra were taken in the reflectron positive ion mode. Spectra were calibrated using the external standards angiotensin II (1046.5 [M+H]1+) and ACTH (2465.2 [M+H]1+). The MALDI-TOF mass spectrometry data were acquired and analyzed using the Bruker XMass software.
Assay Procedures for the Wild-Type and Mutant AST IVs. The enzyme activity during the expression and purification process was monitored using a methylene blue assay procedure described previously (Sheng et al., 2001), with 2-naphthol as substrate at pH 5.5. For determination of kinetic constants, activities of the recombinant AST IV in sulfation of the stereoisomers of 1,2,3,4-tetrahydro-1-naphthol (Sigma-Aldrich) were determined using a published HPLC method for determination of substrate-dependent formation of PAP in the reaction (Duffel et al., 1989; Sheng et al., 2001). In these reactions, assay mixtures of 30-µl total volume contained 0.25 M potassium phosphate buffer (pH 7.0), 8.3 mM 2-mercaptoethanol, 0.2 mM PAPS, and various concentrations of the substrates dissolved in acetone (the final concentration of acetone in the assay was 5% v/v). Reactions were initiated by addition of enzyme (1.0–5.4 µg), incubated at 37°C for 15 min, and terminated by addition of 30 µl of methanol. Aliquots (1.5–3.0 µl) of enzyme solutions containing 0.7 to 4.5 mg/ml of wild-type or mutant AST IV were added by glass microliter syringe to each assay mixture. Concentrations of enzyme in the stock solutions of AST IV and the mutant enzymes were determined by the modified Lowry procedure (Bensadoun and Weinstein, 1976). The concentration of PAP formed in each reaction was determined by HPLC on an Econosphere C18 column (5 µ, 4.6 mm x 250 mm; Alltech Associates, Deerfield, IL). The mobile phase consisted of water/methanol (88:12, v/v) containing 65 mM potassium phosphate, 1.0 mM 1-octylamine, and 65 mM ammonium chloride, at pH of 5.45 before addition of methanol. A flow rate of 2 ml/min and detection at 259 nm were 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. Results from control reaction mixtures (all components present except substrate) were subtracted from the concentrations of PAP formed in complete reaction mixtures to obtain the substrate-dependent formation of PAP.
Calculation of Kinetic Constants. At least six concentrations of each substrate were used, and these included concentrations both greater than and less than the apparent Km value. Apparent Km and Vmax values were obtained by nonlinear least-squares fitting of initial rate data to the equation: V = Vmax · [S]/(Km + [S]). The apparent Km and Vmax values are presented as the mean ± the standard error. The molecular mass of AST IV calculated from the deduced amino acid sequence (33,909) was used for determination of kcat.
Molecular Modeling. Computational studies on 1,2,3,4-tetrahydro-1-naphthol and 2-naphthol in the active site of the rat AST IV were performed using the SYBYL molecular modeling package (SYBYL version 6.7; Tripos Inc., St. Louis, MO) running on a Silicon Graphics O2 workstation (SGI, Mountain View, CA). A previously developed homology model of AST IV (King et al., 2000) was used as the basis for all docking and energy minimization calculations. Coordinates for modeling an estradiol molecule at the sulfuryl acceptor site were obtained from the structure of a mouse estrogen sulfotransferase-17ß-estradiol-PAP complex (PDB crystal structure code: 1AQU
[PDB]
) (Kakuta et al., 1997). The three-dimensional structures of 2-naphthol and each of the enantiomers of 1,2,3,4-tetrahydro-1-naphthol were created with the Build/Edit module of SYBYL and optimized by energy minimization using the Tripos force field. Correct atom types, bond types, and chiral centers were defined with respect to the SYBYL mol2 file format. The structure of each substrate was then prepositioned into the sulfuryl acceptor binding site by aligning the oxygen atom of the hydroxyl group with the position of the 3 oxygen of the 17ß-estradiol that had been determined by homology with the crystal structure of mouse estrogen sulfotransferase. The docking experiments between substrate and protein were conducted manually using the dock module of SYBYL. Calculations of the total energy of each docked substrate/enzyme pair were performed using the SYBYL program at 10-degree intervals of rotation about the carbon-oxygen bond.
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) based on the electron density map of mouse estrogen sulfotransferase (Kakuta et al., 1997). Mouse estrogen sulfotransferase serves as a particularly good structure for homology modeling of AST IV due to the availability of crystal structures that include enzyme-substrate and enzyme-product complexes as well as the structure of an enzyme-PAP-vanadate complex that serves as a transition state model (Kakuta et al., 1998). Moreover, the coordinates of the mouse estrogen sulfotransferase have also been used as the model for determining structure factor phases by molecular replacement for crystal structures of other aryl/phenol sulfotransferases, including human phenol sulfotransferases SULT1A1 (Gamage et al., 2003) and SULT1A3 (Bidwell et al., 1999; Dajani et al., 1999), as well as human estrogen sulfotransferase SULT1E1 (Pedersen et al., 2002). The structure of (R)-(-)-1,2,3,4-tetrahydro-1-naphthol (Figs. 1 and 2) was docked into the homology model for AST IV with the oxygen placed in the position analogous to the oxygen of estradiol in the crystal structure of mouse estrogen sulfotransferase. Although the homology model revealed several amino acid residues in the vicinity of (R)-(-)-1,2,3,4-tetrahydro-1-naphthol, three amino acid residues, Phe77, Phe138, and Tyr236, were identified as occupying positions that could potentially provide steric interactions near the benzylic carbon and aromatic ring of the substrate that might be important to the stereospecificity of the enzyme with the enantiomers of 1,2,3,4-tetrahydro-1-naphthol (Fig. 2).
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Expression and Purification of the Wild-Type and Mutant AST IV Enzymes. We tested the hypothesis generated by the homology modeling studies described above by alanine substitution at residues Phe77, Phe138, and Tyr236. Three bacterial expression vectors for the mutant AST IV sequences, F77A, F138A, and Y236A, were constructed as described under Materials and Methods, and the wild-type and mutant sulfotransferases were overexpressed in E. coli BL21 (DE3) cells. SDS-PAGE and Western blot analysis of cell extracts revealed that the wild type and mutant exhibited identical mobilities and reactivity with antiserum to rat hepatic AST IV (Fig. 3).
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Although a method for bacterial expression and purification of AST IV has been previously described (Chen et al., 1992), a procedure adaptable to expression and rapid purification of sufficient amounts of the three mutant AST IV proteins was required. Therefore, we developed a rapid purification procedure based on PAP-agarose affinity chromatography with subsequent separation of the excess PAP from the purified proteins by gel permeation chromatography. Purifications of wild-type and mutant AST IV proteins are summarized in Table 1. For wild-type AST IV, the yield of purified enzyme from this procedure was 23% based on enzyme units in the cell extract, and yields of the purified F77A, F138A, and Y236A enzymes from cell extracts were 15, 45, and 36%, respectively. Although most of the PAP in the eluting buffer was removed from the sulfotransferase by passing the protein solution through a PD-10 column, a small amount of the PAP (59 µM PAP in a 44 µM solution of AST IV) was not removed by this gel permeation method. This low concentration of PAP did not, however, interfere with the determination of reaction kinetics when the enzyme was diluted in the assay mixture in the presence of saturating concentrations of PAPS.
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Characterization of the Wild-Type and Mutant AST IV Enzymes. As seen in Table 1, all three mutant enzymes catalyzed the sulfation of the standard phenol sulfotransferase substrate, 2-naphthol, at pH 5.5, although the specific activities of the purified mutant enzymes were lower than that seen with the wild-type AST IV. Verification of the expressed protein sequences of these sulfotransferases was accomplished by tryptic digestion and analysis of the peptide fragments by MALDI-TOF mass spectrometry. Mass values of tryptic peptides were matched with the values for the expected sequences of the wild-type and mutant enzymes (11 peptides analyzed from wild-type AST IV represented 50% of the protein sequence; 15 peptides analyzed from F77A represented 77% of the protein sequence; 14 peptides analyzed from F138A represented 64% of the protein sequence; and 15 peptides analyzed from Y236A represented 77% of the protein sequence). The peptides incorporating the regions of site-directed mutation, and their corresponding masses are presented in Table 2.
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Determination of the Catalytic Stereoselectivity of the Wild-Type and Mutant AST IV Enzymes. The purified recombinant enzymes F77A, F138A, and Y236A, as well as the wild-type AST IV, were examined for their stereoselectivity in catalyzing sulfation of the enantiomers of 1,2,3,4-tetrahydro-1-naphthol (Table 3). The overall catalytic efficiencies, as quantified by kcat/Km values, of the F77A, F138A, and Y236A mutants were decreased when compared with wild-type. However, our results showed that the F77A and F138A mutant proteins had catalytic activities with both (R)-(-)- and (S)-(+)-stereoisomers of 1,2,3,4-tetrahydro-1-naphthol, suggesting that Phe77 and Phe138 are residues that are critical in defining the stereochemical properties of AST IV with this chiral substrate. The stereospecificity of the Y236A enzyme, however, was unchanged from the wild-type enzyme, although there was an overall decrease in reaction velocity and catalytic efficiency (Table 3).
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Computational Analysis of the Substrate/Enzyme Interaction. To gain additional insights into the molecular basis of the stereoselectivity of the rat AST IV, the interactions between the enantiomers of 1,2,3,4-tetrahydro-1-naphthol and the AST IV active site were modeled. The bond between the hydroxyl group and its adjacent carbon atom for the 1,2,3,4-tetrahydro-1-naphthol was positioned by alignment with the C3-oxygen bond of 17ß-estradiol that had been modeled into the crystal structure of the AST IV molecular model by homology to a previously described estrogen sulfotransferase-estradiol complex (Kakuta et al., 1997). With the position of the oxygen and adjacent benzylic carbon atom in 1,2,3,4-tetrahydro-1-naphthol fixed, the total energy of the substrate and the active site was calculated after each 10-degree rotation of the molecule around the axis defined by this carbon-oxygen bond. As seen in Fig. 4, the results indicated that major energy barriers to rotation of the tetrahydronaphthols were linked to the presence of the aromatic moieties of Phe77 and Phe138. Removal of either of these aromatic side chains (i.e., conversion from phenylalanine to alanine) allowed additional conformational flexibility within the substrate-binding pocket and altered the enzyme's stereochemical preference for (R)-(-)-tetrahydro-1-naphthol as substrate (Table 3).
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As a further investigation of the loss of catalytic efficiency as a function of removal of steric barriers at the active site of the enzyme, we examined the standard phenol sulfotransferase substrate, 2-naphthol, with the same computational techniques applied to our homology model of AST IV. As seen in Fig. 5, both Phe77 and Phe138 appear to provide barriers to rotation of 2-naphthol in the homology model. Removal of the phenyl ring in either F77A or F138A results in both decreased catalytic function and decreased barriers to substrate rotation (Table 1, Fig. 5). In contrast, Tyr236 does not appear to affect rotation of 2-naphthol in the active site model for the enzyme and has only a minimal effect on catalytic function.
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Discussion |
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One example of investigations where structural information has guided studies on relationships between sulfotransferase structure and substrate specificity involves the catalytic specificity of the mouse estrogen sulfotransferase (mEST or St1e5) for estradiol as opposed to dehydroepiandrosterone (DHEA), a substrate for hydroxysteroid sulfotransferase (Petrotchenko et al., 1999). A combination of crystallographic and mutagenesis techniques was used to determine that tyrosine-81 in mEST prevents efficient binding of dehydroepiandrosterone in the sulfuryl acceptor site of mEST due to steric interactions with C-19 methyl of DHEA (Petrotchenko et al., 1999). These studies showed that mutation of Tyr81 in mEST to a leucine rendered the enzyme capable of catalyzing sulfation of DHEA with kinetic constants similar to those of a wild-type hydroxysteroid sulfotransferase. Moreover, the results of these and further mutational studies on the mEST led to the proposal that Tyr81 and Phe142 in this enzyme form a gate-like structural feature that is important in discrimination between estradiol and DHEA (Petrotchenko et al., 1999).
Structural information has also been coupled with modeling and mutagenesis studies in the investigation of the differences in specificity for catecholamine substrates and 4-nitrophenol that are seen between the human aryl (phenol) sulfotransferase isoforms SULT1A1 (also known as ST1A3 or P-PST) and SULT1A3 (also known as ST1A5 or M-PST). In this case, the enhanced specificity of SULT1A3 for catecholamines relative to 4-nitrophenol and the reverse specificity with SULT1A1 were linked to a glutamate residue (Glu146) in SULT1A3 that corresponds to an alanine in SULT1A1 (Dajani et al., 1998, 1999; Brix et al., 1999). Additional investigations on SULT1A1 and SULT1A3 have indicated that Glu146 as well as residues 84–90 are implicated in the stereospecific sulfation of the D-isomers of DOPA and tyrosine (Pai et al., 2003).
In the present studies we have combined molecular modeling, site-directed mutagenesis, and kinetic analysis to investigate the stereoselectivity of the rat AST IV. The starting point for these studies involved the use of a previously developed homology model for the three-dimensional structure of this phenol sulfotransferase (King et al., 2000), and preliminary molecular docking experiments led to the hypothesis that one or more of three aromatic residues, Phe77, Phe138, or Tyr236, might be important in determining steric interactions with chiral benzylic alcohols leading to stereospecificity. Although the part of the original hypothesis relating to Tyr236 was disproved, the retention of stereospecificity upon alteration of this aromatic residue that is near the docked substrate in the homology model is significant, since it serves as a control experiment that highlights the specificity of the interactions of the other two aromatic residues in this region of the sulfotransferase.
In support of two components of our original hypothesis, the site-directed mutagenesis studies identified Phe77 and Phe138 as essentially equally important in determining the stereospecificity of the AST IV for 1,2,3,4-tetrahydro-1-naphthol. Although the results with Phe77 bear some similarity to the role of the homologous Tyr81 in steric interactions of steroid substrates for estrogen sulfotransferase (Petrotchenko et al., 1999), the comparative roles of Phe138 in AST IV and its homologous Phe142 in estrogen sulfotransferase appear to differ. In estrogen sulfotransferase, Phe142 was a significant residue for the overall catalytic efficiency of estradiol sulfation and was important in forming a gate-like structure in combination with Tyr81, but mutation of Phe142 alone (i.e., leaving Tyr81 intact) altered overall catalytic efficiency but not the specificity of the enzyme for estradiol in relation to dehydroepiandrosterone (Petrotchenko et al., 1999). In the case of the two homologous residues in aryl (phenol) sulfotransferase AST IV, mutation of either Phe77 or Phe138 affects both the stereospecificity and catalytic function of the enzyme with the probe substrate.
Upon further analysis of the kinetic results, although the stereospecificity of the Y236A mutant for sulfation of 1,2,3,4-tetrahydro-1-naphthol isomers was retained, the maximal velocity and catalytic efficiency of the reactions were reduced. Likewise, the rates of the sulfation of 1,2,3,4-tetrahydro-1-naphthol isomers catalyzed by the F77A and F138A sulfotransferases were also diminished. Although significant decreases in reaction velocity were seen with the phenolic substrate, 2-naphthol, for the F77A and F138A mutant enzymes, the rate of reaction with 2-naphthol catalyzed by the Y236A mutant compared much more favorably with the wild-type AST IV. The relatively small change in reaction velocity for sulfation of 2-naphthol catalyzed by the Y236A mutant suggests that major structural changes in the overall structure of the enzyme have not occurred. Thus, differences in rates of reaction and catalytic efficiency appear to be due primarily to structural interactions of the individual molecules with amino acids at the sulfuryl acceptor site rather than general changes in overall structure of the protein. These differences in the stereospecificity observed with F77A and F138A led to further modeling of the interaction of 1,2,3,4-tetrahydro-1-naphthol with the active sites of the wild-type and mutant enzymes in the homology model.
We were able to obtain an approximation of the role of steric effects on the orientation of substrates through a systematic probe of 2-naphthol and each of the two enantiomers of 1,2,3,4-tetrahydronaphthol docked in the active site model. For this study, the hydroxyl group and adjacent carbon atom in the sulfuryl acceptor were positioned in the homology model by analogy to the crystal structures of catalytic complexes that were previously determined for 17ß-estradiol and estrogen sulfotransferase (Kakuta et al., 1997, 1998). While holding the coordinates of those two atoms fixed in the model of the active site, the total energy of the system was calculated after each 10-degree rotation around the carbon-oxygen bond of the sulfuryl acceptor. This produced a map of interactions at the active site, with the changes due to site-directed mutagenesis showing up as a difference from the wild-type enzyme in calculated energy profile.
Comparison of the overall catalytic efficiencies of the wild-type and mutant enzymes with the relative profiles of interactions obtained from the models clearly shows that removal of aromatic side chains from F77 and F138 at the sulfuryl acceptor site (i.e., mutations of phenylalanine to alanine) results in lower catalytic efficiency for the enantiomeric 1,2,3,4-tetrahydronaphthols. Although one might interpret this as due to a lowered ability to facilitate the initial binding of the molecule in a catalytically competent enzyme-substrate complex, it may also be a result of the hindered conformational rotation of the molecule in the active site to ultimately reach an orientation of the molecule such that sulfuryl transfer may occur. Thus, removal of barriers either to initial binding or to subsequent conformational change of the substrate, as seen with the F77A and F138A mutants, leads to the ability of the enzyme to accept both enantiomers of 1,2,3,4-tetrahydronaphthol as substrates, but this comes at the expense of decreased catalytic efficiency.
Although these results on interactions at the active site of AST IV based on homology models and calculated relative energies are consistent with interpretations of the importance of active site geometry in substrate recognition, they clearly represent an early attempt at modeling these interactions. In addition to providing a basis for further experimental probes of specificity in aryl (phenol) sulfotransferases, they also serve as a starting point for the more extensive modeling studies necessary for exploration of the role of conformational changes by both substrate and enzyme in determining molecular specificity of the sulfotransferases. Indeed, since recent studies suggest that the human phenol sulfotransferase SULT1A1 may adopt structural conformations not seen in the crystal structure to accommodate estradiol as substrate (Gamage et al., 2003), it will be important to learn the extent to which the rat AST IV might exhibit similar conformational changes. Additionally, the possibility that these mutant forms of AST IV might actually facilitate binding and catalysis with larger substrates also remains to be explored.
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Footnotes |
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1 Abbreviations used are: PAPS, 3'-phosphoadenosine 5'-phosphosulfate; AST IV, rat aryl sulfotransferase IV or tyrosine ester sulfotransferase (GenBank accession number: X52883
[GenBank]
; SWISS-PROT: P17988
[GenBank]
); PAP, adenosine 3', 5'-diphosphate; PAGE, polyacrylamide gel electrophoresis; MALDI-TOF, matrix-assisted laser desorption ionization/time-of-flight; HPLC, high-pressure liquid chromatography; mEST, mouse estrogen sulfotransferase (GenBank accession number: S78182
[GenBank]
; PDB codes: 1AQU
[PDB]
, 1AQY
[PDB]
); DHEA, dehydroepiandrosterone. 
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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