Sulfuryl Group Transfer Research Articles

Fluorinated Phosphoadenosine 5'-Phosphosulfate Analogues for Continuous Sulfotransferase Activity Monitoring and Inhibitor Screening by 19F NMR Spectroscopy.

Sulfotransferases (STs) are ubiquitous enzymes that participate in a vast number of biological processes involving sulfuryl group (SO3) transfer. 3′-phosphoadenosine 5′-phosphosulfate (PAPS) is the universal ST cofactor, serving as the “active sulfate” source in cells. Herein, we report the synthesis of three fluorinated PAPS analogues that bear fluorine or trifluoromethyl substituents at the C2 or C8 positions of adenine and their evaluation as substitute cofactors that enable ST activity to be quantified and real-time-monitored by fluorine-19 nuclear magnetic resonance (19F NMR) spectroscopy. Using plant AtSOT18 and human SULT1A3 as two model enzymes, we reveal that the fluorinated PAPS analogues show complementary properties with regard to recognition by enzymes and the working 19F NMR pH range and are attractive versatile tools for studying STs. Finally, we developed an 19F NMR assay for screening potential inhibitors against SULT1A3, thereby highlighting the possible use of fluorinated PAPS analogues for the discovery of drugs for ST-related diseases.

A Sulfuryl Group Transfer Strategy to Selectively Prepare Sulfated Steroids and Isotopically Labelled Derivatives.

The treatment of common steroids: estrone, estradiol, cortisol, and pregnenolone with tributylsulfoammonium betaine (TBSAB) provides a convenient chemoselective conversion of the steroids alcohol/phenol moiety to the corresponding steroidal organosulfate. An important feature of the disclosed methodology is the millimolar scale of the reaction, and the isolation of the corresponding steroid sulfates as their biologically relevant sodium salts without the need for ion-exchange chromatography. The scope of the method was further explored in the estradiol and pregnanediol steroid systems with the bis-sulfated derivatives. Ultimately, a method to install an isotopic label, deuterium (2H) combined with estrone sulfation is a valuable tool for its mass-spectrometric quantification in biological studies.

Apoptosis inducing anthraquinone rhein and emodin differentially suppress human dehydroepiandrosterone sulfotransferase (hSULT2A1) and phenol sulfotransferases (hSULT1A1) in Hep-G2 and Caco-2 cells

The anti-cancer and apoptosis-inducing drugs rhein (4, 5-dihydroxyanthraquinone-2-carboxylic acid) and emodin (3methyl-1, 6, 8-trihydro-xyanthrax-quinone) are clinically very important. They modulate cell cycle via tumor suppressor gene, immuno-receptors and ligand activated nuclear receptors. Our recent observation suggests for the first time that 10 days of treatment of either drug with various concentrations (0.01 to 100 M) differentially suppressed the sulfotransferases (SULTs) activities and protein expressions in human hepatocellular carcinoma (Hep G2) and intestinal carcinoma (Caco-2) cell lines. SULTs are phase II drug metabolizing enzymes which catalyze the sulfuryl group transfer to hydroxyl containing endobiotics and xenobiotics. In the present investigation, dehydroepiandrosterone SULT (hSULT1A1) was markedly suppressed by these drugs in human cells. This is the first time report which demonstrates that rhein and emodin may regulate human SULTs. Our finding has important physiological and clinical implications. It will help in the understanding of the SULTs regulations by clinically important drugs and xenobiotics. In future, these drugs may be used in a better defined manner, taking into account its SULTs suppression effects with possible physiological consequences.

Linear correlation between effective charge and bond length sensitivity to electronic effects in phosphoryl, sulfonyl, and sulfuryl compounds

This work presents a linear correlation between bond length sensitivity (Δr/ΔpKlg) and effective charge by comparing a series of the following ROX compounds bearing phosphoryl (XP) and sulfonyl and sulfuryl (XS) moieties: phosphate monoester dianions and monoanions, phosphate diesters, phosphate and phosphorothioate triesters, 4‐Me, 3‐NO2 and 2‐ or 4‐NO2 substituted aryl benzene sulfonates, sulfate monoesters, and a previously determined series of sulfamate and mesylate esters. In every series for ROX compounds, the shortest C―O bond corresponds to the longest O―X bond, showing a linear correlation with the pKlg of the ROH parent compound. The chemical reasons for the extent of bond elongation are discussed, and a linear correlation is found between O―X bond length sensitivity and effective charge. These data show that bond length elongation depends on its strength regarding the equilibrium related to the full bond cleavage. Effective charges for phosphorothioate triesters and sulfonates are estimated, and the effective charge for aryl sulfamate esters is determined on the basis of the available linear free energy relationship data. It is expected that the observations of this paper will illuminate the effect of the electronic demand on the O―X bond lengths during phosphoryl, sulfonyl, and sulfuryl group transfer. Copyright © 2013 John Wiley & Sons, Ltd.

Fluorescence assay for protein post-translational tyrosine sulfation

We developed a fluorescent assay to conveniently determine the kinetics of protein sulfation, which is essential for understanding interface between protein sulfation and protein-protein interactions. Tyrosylprotein sulfotransferase (TPST) catalyzes protein sulfation using 3'-phosphate 5'-phosphosulfate (PAPS) as sulfuryl group donor. In this report, PAPS was regenerated following sulfuryl group transfer between adenosine 3',5'-diphosphate and 4-methylumbelliferyl sulfate catalyzed by phenol sulfotransferase (PST). The TPST and PST coupled enzyme platform continuously generated fluorescent 4-methylumbelliferone (MU) that was used to real-time monitor protein sulfation. Using a recombinant N utilization substance protein A fused Drosophila melanogaster tyrosylprotein sulfotransferase, we demonstrated that the activity of TPST determined through MU fluorescence directly correlated with protein sulfation. Kinetic constants obtained with small P-selectin glycoprotein ligand-1 peptide (PSGL-1 peptide, MW 1541) or its large glutathione S-transferase fusion protein (GST-PSGL-1, MW 27833) exhibited significant variation. This assay can be further developed to a high-throughput method for the characterization of TPSTs and for the identification and screening of their protein substrates.

Kinetic measurements and mechanism determination of Stf0 sulfotransferase using mass spectrometry

Mycobacterial carbohydrate sulfotransferase Stf0 catalyzes the sulfuryl group transfer from 3′-phosphoadenosine-5′-phosphosulfate (PAPS) to trehalose. The sulfation of trehalose is required for the biosynthesis of sulfolipid-1, the most abundant sulfated metabolite found in Mycobacterium tuberculosis. In this paper, an efficient enzyme kinetics assay for Stf0 using electrospray ionization (ESI) mass spectrometry is presented. The kinetic constants of Stf0 were measured, and the catalytic mechanism of the sulfuryl group transfer reaction was investigated in initial rate kinetics and product inhibition experiments. In addition, Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry was employed to detect the noncovalent complexes, the Stf0–PAPS and Stf0–trehalose binary complexes, and a Stf0–3′-phosphoadenosine 5′-phosphate–trehalose ternary complex. The results from our study strongly suggest a rapid equilibrium random sequential Bi-Bi mechanism for Stf0 with formation of a ternary complex intermediate. In this mechanism, PAPS and trehalose bind and their products are released in random fashion. To our knowledge, this is the first detailed mechanistic data reported for Stf0, which further demonstrates the power of mass spectrometry in elucidating the reaction pathway and catalytic mechanism of promising enzymatic systems.

Arginine Residues in the Active Site of Human Phenol Sulfotransferase (SULT1A1)

Cytosolic sulfotransferases (STs) catalyze the sulfation of hydroxyl containing compounds. Human phenol sulfotransferase (SULT1A1) is the major human ST that catalyzes the sulfation of simple phenols. Because of its broad substrate specificity and lack of endogenous substrates, the biological function of SULT1A1 is believed to be an important detoxification enzyme. In this report, amino acid modification, computer structure modeling, and site-directed mutagenesis were used for studies of Arg residues in the active site of SULT1A1. The Arg-specific modification reagent, 2,3-butanedione, inactivated SULT1A1 in an efficient, time- and concentration-dependent manner, suggesting Arg residues play an important role in the catalytic activity of SULT1A1. According to the computer model, Arg78, Arg130, and Arg257 may be important for SULT1A1 catalytic activity. Site-directed mutagenesis results demonstrated that the positive charge on Arg78 is not critical for SULT1A1 because R78A is still active. In contrast, a negative charge at this position, R78E, completely inactivated SULT1A1. Arg78 is in close proximity to the site of sulfuryl group transfer. Arg257 is located very close to the 3'-phosphate in adenosine 3'-phosphate 5'-phosphosulfate (PAPS). Site-directed mutagenesis demonstrated that Arg257 is critical for SULT1A1: both R257A and R257E are inactive. Although Arg130 is also located very close to the 3'-phosphate of PAPS, R130A and R130E are still active, suggesting that Arg130 is not a critical residue for the catalytic activity of SULT1A1. Computer modeling suggests that the ionic interaction between the positive charge on Arg257, and the negative charge on 3'-phosphate is the primary force stabilizing the specific binding of PAPS.

Modeling the Binding and Inhibition Mechanism of Nucleotides and Sulfotransferase Using Molecular Docking

AbstractPhenol sulfotransferase (PST) catalyzes sulfuryl group transfer from adenosine 3′‐phosphate 5′‐phosphosulfate (PAPS) to a variety of nucleophilic acceptors in biological systems. Physiological sulfation by PAPS results in the production of adenosine 3′,5′‐diphosphate (PAP). PAP may become a strong inhibitor or cofactor for the physiological or the transfer reactions, respectively. Several nucleotides other than PAP also serve as substrates, inhibitors or cofactors of PST catalyzed reactions. We are interested in the effects of these nucleotides on the PST catalyzed reactions and their possible physiological significance in biology. Several nucleotide inhibitors (such as adenosine 5′‐diphosphate and adenosine 5′‐triphosphate) of sulfotransferase in physiological reactions have been reported in the literature. However, our data suggests that they are not inhibitors for the transfer reaction or the reverse physiological reaction catalyzed by PST. The aim of this work is to show that the molecular docking analysis can be successfully used to underline the inhibition mechanism of these nucleotides. First, the selected compounds were subjected to a detailed docking analysis, by means of GEMDOCK, a program able to reveal the most likely binding mode for each ligand. By comparing these results with binding sites and binding compounds of the nucleotides with known X‐ray structures, it is possible to highlight the site specificity and the inhibition mechanism of the select compounds. The results obtained by the above algorithm were further confirmed experimentally. In this paper, we show the effect of a variety of nucleotides on the activity of sulfotransferase in different docking conditions.

Mechanism of posttranslational regulation of phenol sulfotransferase: expression of two enzyme forms through redox modification and nucleotide binding.

Sulfotransferase catalyzes sulfuryl group transfer between a nucleotide and a variety of nucleophiles that may be sugar, protein, xenobiotics, and other small molecules. Nucleotides may serve as cosubstrate, cofactor, inhibitor, or regulator in an enzyme catalyzed sulfuryl group transfer reaction. We are trying to understand how nucleotide regulates the activity of phenol sulfotransferase (PST) through the expression of two enzyme forms. The homogeneous rat recombinant PST was obtained from Escherichia coli, and the nucleotide copurified was examined. The nucleotide was completely removed from inactive PST in high salt and oxidative condition. Total enzyme activity was recovered following incubation in reductive environment. Many nucleotides are known to tightly bind to PST but only one nucleotide, 3'-phosphoadenosine 5'-phosphate (PAP), was identified to combine with PST by ion-pair RP-HPLC, UV-visible spectra, (31)P NMR, and ESI-MS and MS-MS spectrometry. In addition to the presence or absence of PAP, oxidation following reduction of PST was required to completely interconvert the two forms of PST. According to the experimental results, a mechanism for the formation of the two enzyme forms was proposed.

Spectrofluorimetric analysis of 7-hydroxycoumarin binding to bovine phenol sulfotransferase

Phenol sulfotransferases (SULT1s, EC 2.8.2.1) catalyze sulfuryl group transfer from 3′-phosphoadenosine-5′-phosphosulfate (PAPS) to the hydroxyl oxygen of aromatic acceptor substrates. Previous work with the bovine SULT1A1 has utilized the highly fluorescent substrate 7-hydroxycoumarin (7-HC, umbelliferone) as an acceptor substrate [Biochem. Biophys. Res. Commun. 261 (1999) 815]. Here we report that adenosine-3′,5′-bisphosphate (PAP)-dependent binding of 7-HC to bSULT1A1 can be observed due to the appearance of a 400–420-nm shoulder in the emission spectrum, using an excitation wavelength of 280 nm. This emission was observed by placing 7-HC in ethanol, which is consistent with bSULT1A1 phenol binding site hydrophobicity. Titrations with 7-HC indicate a K d for 7-HC of 0.58 μM and substoichiometric binding to the homodimeric enzyme. The bSULT1A1:PAP:7-HC complex could be disrupted with pentachlorophenol (PCP), titrations with which indicated 0.5 equivalents per enzyme subunit. Titrations of enzyme plus 7-HC with PAP also indicated 0.5 equivalents per enzyme subunit. These results suggest a model of homodimeric bSULT1A1 in which subunit interactions favor half-site reactivity in the formation of a dead end complex.

Comparative molecular field analysis of substrates for an aryl sulfotransferase based on catalytic mechanism and protein homology modeling.

Comparative Molecular Field Analysis (CoMFA) methods were used to produce a 3D-QSAR model that correlated the catalytic efficiency of rat hepatic aryl sulfotransferase (AST) IV, expressed as log(k(cat)/K(m)), with the molecular structures of its substrates. A total of 35 substrate molecules were used to construct a CoMFA model that was evaluated on the basis of its leave-one-out cross-validated partial least-squares value (q(2)) and its ability to predict the activity of six additional substrates not used in the training set. The model was constructed using substrate conformations that favored (1) proton abstraction by the catalytic histidine residue, (2) an in-line sulfuryl-group transfer mechanism, and (3) constraints imposed by the residues lining the substrate binding pocket of a homology model of AST IV. This CoMFA model had a q(2) value of 0.691, and it successfully predicted the activities of the six molecules not used in the training set. A final CoMFA model was constructed using the same methodology but with molecules from both the training set and the test set. Its q(2) value was 0.701, and it had a non-cross-validated r(2) value of 0.922. The contour coefficient map generated by this CoMFA was overlaid on the amino acids in the substrate-binding pocket of the homology model of AST IV and found to show a good fit. Additionally external validation was obtained by using the CoMFA model to design substrates that show high activities. These results establish a methodology for prediction of the substrate specificity of this sulfotransferase based on CoMFA methods that are guided by both the homology model and the catalytic mechanism of the enzyme.

Kinetic analysis of NodST sulfotransferase using an electrospray ionization mass spectrometry assay.

A novel and efficient enzyme kinetics assay using electrospray ionization mass spectrometry was developed and applied to the bacterial carbohydrate sulfotransferase (NodST). NodST catalyzes the sulfuryl group transfer from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to chitobiose, generating 3'-phosphoadenosine 5'-phosphate (PAP) and chitobiose-6-OSO(3)(-) as products. Traditional spectrophotometric assays are not applicable to the NodST system since no shift in absorption accompanies sulfuryl group transfer. Alternative assays have employed thin-layer chromatography, but this procedure is time-consuming and requires radioactive materials. The ESI-MS assay presented herein requires no chromophoric substrate or product, and the analysis time is very short. The ESI-MS assay is used to determine NodST kinetic parameters, including K(M), V(max), and K(i) (for PAP). In addition, the mode of inhibition for PAP was rapidly determined. The results were in excellent agreement with those obtained from previous assays, verifying the accuracy and reliability of the ESI-MS assay. This unique technique is currently being used to investigate the enzymatic mechanism of NodST and to identify sulfotransferase inhibitors.

Nucleotide Binding and Sulfation Catalyzed by Phenol Sulfotransferase

The sulfation of a nucleotide is an indispensable step for the sulfuryl group transfer in a biological system. The product and cosubstrate of sulfotransferase in physiological condition are adenosine 3′,5′-bisphosphate (PAP) and 3′-phospho adenosine 5′-phosphosulfate (PAPS), respectively. We find that ribose and adenine, two major parts of the adenosine nucleotide, bind tightly to phenol sulfotransferase (PST) separately, and various nucleotides also bind tightly to PST. We determine the dissociation constants of a variety of nucleotides and examine their potential as cofactors or cosubstrates of PST. Using 4-nitrophenyl sulfate as the sulfuryl group donor, three nucleotides, adenosine 5′-monophosphate (AMP), adenosine 2′,5′-bisphosphate (2′,5′-PAP), and adenosine 2′:3′-cyclic phosphate 5′-phosphate (2′:3′-cyclic PAP), are shown here for the first time to be sulfated at 5′-phopho position by a PST catalyzed reaction. Spectrophotometry, HPLC, and 31P NMR are used to determine the activity of PST and identify the sulfated nucleotides. The Vmax of PST and Km of these nucleotides are determined when they are used as cofactors or cosubstrates for the sulfuryl group transfer. The existence and possible physiological significance of these newly reported binding and sulfation of nucleotides by PST in biology is yet to be discovered.

A quantum mechanical study of the transfer of biological sulfate

The biological process of enzymatic sulfuryl group transfer has been studied by ab initio (density-functional and Hartree–Fock) and semiempirical quantum mechanical methods. The active site of estrogen sulfotransferase in ternary complex with a sulfate donor (PAPS) and sulfate acceptor (estradiol) is modeled. The mechanism proposed in a recent X-ray crystal structure paper (Kakuta et al., Nat. Struct. Biol. 4 (1997) 904) serves as the basis for the calculations. We find that the mechanism proposed in the crystallographic paper is reasonable. The sulfonation takes place in several key steps: neutralization of the charge on PAPS, lengthening of the bridging S–O bond with no cost in energy, activation of the attacking oxygen and proton transfer from estradiol to histidine and then to the sulfuryl group.

Colorimetric Determination of the Purity of 3′-Phospho Adenosine 5′-Phosphosulfate and Natural Abundance of 3′-Phospho Adenosine 5′-Phosphate at Picomole Quantities

This work presents novel colorimetric methods not only to measure 3′-phospho adenosine 5′-phosphate (PAP) and 3′-phospho adenosine 5′-phosphosulfate (PAPS) in the range of picomoles, but also to determine the purity of PAPS or PAP contaminants in PAPS in the range of nanomoles. These methods exploit the availability of overexpressed phenol sulfotransferase (PST) and the fact that sulfuryl group transfer requires the use of PAP or PAPS as a cofactor or cosubstrate. Experimental results indicate that absorption at 400 nm due to the production of 4-nitrophenol (pNP) is catalyzed by PST when the sulfuryl group transfers from 4-nitrophenylsulfate (pNPS) to PAP or to 2-napthol. In the absence of an acceptor substrate, PAPS is hydrolyzed to PAP by PST and is determined by sulfation with pNPS before and after this reaction. The change of absorption of pNP at 400 nm corresponds to the amount of PAP that is hydrolyzed from PAPS. Moreover, a standard curve is constructed using authentic PAP and PAP-free PST. Furthermore, this curve is used to determine the amount of PAP in extracts of pig liver, rat liver, andEscherichia coli.

Two Phenol Sulfotransferase Species from One cDNA: Nature of the Differences

A phenol sulfotransferase from rat liver (EC 2.8.2.9), expressed inEscherichia colifrom a single cDNA, was purified as two separable but catalytically active proteins. The proteins appeared to be identical to each other and to the natural liver sulfotransferase by comparison of their amino acid constitution, amino-terminal end group, and interaction with a polyclonal antibody raised against the liver enzyme. Each of the recombinant forms, α and β, catalyzed the sulfuryl group transfer from 4-nitrophenylsulfate to an acceptor phenol, a reaction in which 3′-phospho-adenosine 5′-phosphate (PAP) is a necessary intermediate. Only form β, however, catalyzes the physiological transfer of a sulfuryl group from 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to the free phenol. Evidence is presented that sulfotransferase α, but not β, has 1 mol of PAP tightly bound per enzyme dimer. The ability to utilize PAPS as a sulfate donor could be altered: form α could be treated and purified as form β to acquire the ability to use PAPS, whereas form β was treated by extended incubation with PAP, lost its ability to use PAPS, and was purified as form α.

Mechanisms of Reaction of Sulfate Esters: A Molecular Orbital Study of Associative Sulfuryl Group Transfer, Intramolecular Migration, and Pseudorotation

Molecular orbital calculations for the reactions of 2-hydroxyethyl sulfate in neutral and ionized forms were used to examine the free energy profiles for intramolecular sulfuryl group transfer in the gas phase. Detailed analysis of reaction dynamics yielded structures for trigonal bipyramidal pentacoordinate reaction intermediates and for transition states at the MP2/6-31+G*//HF/3-21+ G(*) and MP2/6-31+G*//HF/3-21G(*) levels of calculation for anionic and neutral species, respectively. Application of a continuum dielectric model to these structures provided estimated free energy profiles for reaction in aqueous solution. Comparison with the reactivity of phosphate esters suggests that the empirical guidelines for reactions of phosphates may, in large part, be extended to reactions of sulfate esters. It is predicted that the activation barriers to intramolecular sulfuryl group transfer will be large, in accord with experiment and in contrast with phosphoryl transfer. The explanation is based upon (1) the requirement for the energetically unfavorable protonation of the sulfate moiety for nucleophilic attack on sulfur; and (2) the relatively higher energy of the pentacoordinate relative to the tetracoordinate state for S over P species. However, Berry pseudorotation is facile and does not present a barrier to reaction. The calculations suggest that nucleophilic substitution of alkoxide on the protonated sulfate moiety has a low activation barrier and may occur with inversion or retention of stereochemistry at sulfur.

Amine N-sulfotransferase.

A highly purified amine N-sulfotransferase has been isolated from guinea pig liver that catalyzes sulfuryl group transfer from 3'-phosphoadenosine 5'-phosphosulfate to one of a large number of either primary or secondary amines forming the appropriate sulfamate and adenosine 3',5'-bisphosphate. Amines as different as aniline, 2-naphthylamine, octylamine, 1,2,3,4-tetrahydroisoquinoline and 1,2,3,4-tetrahydroisoquinoline, desmethylimipramine, and cyclohexylamine serve as acceptors; the product of the last of these substrates is the sugar-substitute cyclamate. Amine N-sulfotransferase activity is dependent on the presence of an unprotonated amino group. The purified enzyme preparation also has O-sulfotransferase activities, suggesting that transfer to oxygen could represent an intrinsic function of the N-sulfotransferase.

ChemInform Abstract: Bonding in Phosphoryl (‐PO32‐) and Sulfuryl (‐SO32‐) Group Transfer Between Nitrogen Nucleophiles as Determined from Rate Constants for Identity Reactions.

ChemInform Abstract: Bonding in Phosphoryl (‐PO₃^2‐) and Sulfuryl (‐SO₃^2‐) Group Transfer Between Nitrogen Nucleophiles as Determined from Rate Constants for Identity Reactions.

Bonding in phosphoryl (-PO32-) and sulfuryl (-SO3-) group transfer between nitrogen nucleophiles as determined from rate constants for identity reactions

Determination des constantes de vitesse des reactions bimoleculaires de pyridines avec des pyridinophosphonates et pyridinosulfonates dans des solutions aqueuses