Crystal structure-based studies of cytosolic sulfotransferase

Sulfation is a widely observed biological reaction conserved from bacterium to human that plays a key role in various biological processes such as growth, development, and defense against adversities. Deficiencies due to the lack of the ubiquitous sulfate donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS) are lethal in humans. A large group of enzymes called sulfotransferases catalyze the transfer reaction of sulfuryl group of PAPS to the acceptor group of numerous biochemical and xenochemical substrates. Four X-ray crystal structures of sulfotransferases have now been determined: cytosolic estrogen, hydroxysteroid, aryl sulfotransferases, and a sulfotransferase domain of the Golgi-membrane heparan sulfate N-deacetylase/N-sulfotransferase 1. These have revealed the conserved core structure of the PAPS binding site, a common reaction mechanism, and some information concerning the substrate specificity. These crystal structures introduce a new era of the study of the sulfotransferases.     

Paper disk assay for glycosaminoglycan sulfotransferases

A method is described for the assay of sulfotransferases, which transfer sulfate from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to glycosaminoglycan acceptors. Following the sulfation reactions, the [35S]sulfate-labeled products are precipitated and then separated from a sulfate donor ([35S]PAPS) and its degradation products by a paper disk method, and then the radioactivity remaining on the paper disk is subsequently determined by liquid scintillation counting. The rapidity and simplicity of the method are advantageous for multiple assays and have allowed us to establish assay conditions for serum sulfotransferases which introduce sulfate at position 6 of the internal N-acetylgalactosamine units of chondroitin, position 2 (amino group) of the glucosamine units of heparan sulfate and sugar units of keratan sulfate, respectively. The assay method will be applicable with modification to the assay of other glycosaminoglycan sulfotransferases and glycoprotein sulfotransferases.     

Crystal structure of the human estrogen sulfotransferase-PAPS complex: evidence for catalytic role of Ser137 in the sulfuryl transfer reaction

Estrogen sulfotransferase (EST) transfers the sulfate group from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to estrogenic steroids. Here we report the crystal structure of human EST (hEST) in the context of the V269E mutant-PAPS complex, which is the first structure containing the active sulfate donor for any sulfotransferase. Superimposing this structure with the crystal structure of hEST in complex with the donor product 3'-phosphoadenosine 5'-phosphate (PAP) and the acceptor substrate 17beta-estradiol, the ternary structure with the PAPS and estradiol molecule, is modeled. These structures have now provided a more complete view of the S(N)2-like in-line displacement reaction catalyzed by sulfotransferases. In the PAPS-bound structure, the side chain nitrogen of the catalytic Lys(47) interacts with the side chain hydroxyl of Ser(137) and not with the bridging oxygen between the 5'-phosphate and sulfate groups of the PAPS molecule as is seen in the PAP-bound structures. This conformational change of the side chain nitrogen indicates that the interaction of Lys(47) with Ser(137) may regulate PAPS hydrolysis in the absences of an acceptor substrate. Supporting the structural data, the mutations of Ser(137) to cysteine and alanine decrease gradually k(cat) for PAPS hydrolysis and transfer activity. Thus, Ser(137) appears to play an important role in regulating the side chain interaction of Lys(47) with the bridging oxygen between the 5'-phosphate and the sulfate of PAPS.     

Structure and function of sulfotransferases

Sulfotransferases (STs) catalyze the transfer reaction of the sulfate group from the ubiquitous donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to an acceptor group of numerous substrates. This reaction, often referred to as sulfuryl transfer, sulfation, or sulfonation, is widely observed from bacteria to humans and plays a key role in various biological processes such as cell communication, growth and development, and defense. The cytosolic STs sulfate small molecules such as steroids, bioamines, and therapeutic drugs, while the Golgi-membrane counterparts sulfate large molecules including glucosaminylglycans and proteins. We have now solved the X-ray crystal structures of four cytosolic and one membrane ST. All five STs are globular proteins composed of a single alpha/beta domain with the characteristic five-stranded beta-sheet. The beta-sheet constitutes the core of the Paps-binding and catalytic sites. Structural analysis of the PAPS-, PAP-, substrate-, and/or orthovanadate (VO(3-)(4))-bound enzymes has also revealed the common molecular mechanism of the transfer reaction catalyzed by sulfotransferses. The X-ray crystal structures have opened a new era for the study of sulfotransferases.     

Protamine increases the affinity of 3'-phosphoadenosine 5'-phosphosulfate toward a sulfotransferase from chicken embryo epiphyseal cartilage

A 3' -phosphoadenosine 5' -phosphosulfate (PAPS):chondroitin sulfate sulfotransferase from chicken embryo epiphyseal cartilage, which was partially purified, exhibited a molecular mass of 150 kDa. The enzymatic sulfation of totally desulfated chondroitin was activated up to 12-fold by protamine while the sulfation of partially sulfated chondroitin was activated only 3-fold. Protamine increased the affinity of the enzyme for PAPS about 4-fold when partially desulfated chondroitin was used as sulfate acceptor. The S 0.5 for the totally desulfated chondroitin was not affected by protamine, while high PAPS concentration slightly increased the affinity of the enzyme for the same sulfate acceptor. The possible role of these substances in the regulation of the sulfation of chondroitin sulfate is discussed.     

Endocrine steroid sulfotransferases: porcine endometrial estrogen sulfotransferase

Porcine endometrial estrogen sulfotransferase has been isolated and its properties examined. This enzyme only appeared in uteri from ovariectomized gilts which had been primed with estrogen and treated with progesterone. The most stable form of the enzyme was obtained via chromatofocusing of the 100,000 g supernatant from secretory endometrium. A molecular weight of 31 KDa was determined for this sulfotransferase by molecular sieve (Sephadex G-200 Superfine) and disk-gel electrophoresis. The active protein displayed a pI of 6.1, pH optimum of 7.6-7.8 and a requirement of 10 mM Mg2+ for maximum transfer of sulfate from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to estrone (E1). Km of the reaction was 24 +/- 4.7 microM for PAPS and 24 +/- 9.8 nM for E1 as substrate. Porcine endometrial sulfotransferase thus displayed a much greater affinity for E1 than a similar enzyme previously isolated from bovine adrenals. As has been observed of sulfotransferases from other tissues, an endogenous substrate (presumed to be E1) accompanies the enzyme throughout its purification.     

Partial purification and characterization of 3'-phosphoadenylylsulfate:keratan sulfate sulfotransferases

Two 3'-phosphoadenylylsulfate:keratan sulfate sulfotransferases were purified 600-fold and 340-fold, respectively, from isolated bovine cornea cells. Sulfotransferase I exhibited an apparent Mr = 220,000, whereas an Mr = 140,000 was calculated for sulfotransferase II. The final preparations were both devoid of chondroitin sulfate sulfotransferase activity. The position of sulfation was determined by proton nuclear magnetic resonance spectroscopy. Sixty per cent of the sulfate ester groups formed by sulfotransferase I were linked to the C-6 atom of galactosyl residues, the other ones to the C-6 atom of N-acetylglucosamine. Sulfotransferase II showed a different specificity: 23% of the newly formed sulfate ester groups were on galactosyl and 77% on N-acetylglucosaminyl residues. Both sulfotransferase preparations acted in a cooperative manner. In the presence of both sulfotransferases, the incorporation of [35S]sulfate into keratan sulfate was up to 75% higher than could be expected from the sum of individual activities. From the specific radioactivities of the oligosaccharides produced by digestion with endo-beta-galactosidase, it was also concluded that both enzyme species reacted best with keratan sulfate segments exhibiting a relatively high degree of sulfation.     

Sulfation of p-nitrophenyl-N-acetyl-beta-D-galactosaminide with a microsomal fraction from cultured chondrocytes

Chick embryo chondrocyte microsomes containing intact Golgi vesicles took up 3'-phosphoadenosine-5'-phospho[35S]sulfate ([35S]PAPS) in a time- and temperature-dependent, substrate-saturable manner. When [35S]PAPS and p-nitrophenyl-N-acetyl-beta-D-galactosaminide (pNP-GalNAc) were added to the incubation in the absence of detergent, the microsomes catalyzed the transfer of sulfate from [35S]PAPS to pNP-GalNAc to form pNP-GalNAc-6-35SO4. The apparent Km values for PAPS in the uptake and the pNP-GalNAc sulfation reactions were 2 X 10(-7) and 2 X 10(-6) M, respectively. The sulfation of pNP-GalNAc by the microsomal preparation was inhibited by detergent. The microsomal fraction also catalyzed the transfer of sulfate from [35S]PAPS to oligosaccharides prepared from chondroitin. However, in contrast to the sulfation of pNP-GalNAc, the rate of sulfation of these oligosaccharides was low in the absence of detergent and was markedly stimulated when detergent was added. Sulfation of pNP-GalNAc by the freeze-thawed microsomes was inhibited when the octasaccharide prepared from chondroitin was present in the reaction mixture. As the PAPS that had been internalized in the microsomal vesicles was consumed in the sulfation of pNP-GalNAc, more [35S]PAPS was taken up and the sulfated pNP-GalNAc was released from the vesicles. These observations suggest that pNP-GalNAc may serve as a model membrane-permeable substrate for study of the 6-sulfo-transferase reaction involved in sulfation of chondroitin sulfate in intact Golgi vesicles.     

Antioxidant properties of diorganoyl diselenides and ditellurides: modulation by organic aryl or naphthyl moiety

Sulfotransferases catalyze the sulfate conjugation of a wide variety of endogenous and exogenous molecules. Human pathogenic mycobacteria produce numerous sulfated molecules including sulfolipids which are well related to the virulence of several strains. The genome of Mycobacterium avium encodes eight putative sulfotransferases (stf1, stf4-stf10). Among them, STF9 shows higher similarity to human heparan sulfate 3-O-sulfotransferase isoforms than to the bacterial STs. Here, we determined the crystal structure of sulfotransferase STF9 in complex with a sulfate ion and palmitic acid at a resolution of 2.6 ?. STF9 has a spherical structure utilizing the classical sulfotransferase fold. STF9 exclusively possesses three N-terminal α-helices (α1, α2, α3) parallel to the 3'-phosphoadenosine-5'-phosphosulfate (PAPS) binding motif. The sulfate ion binds to the PAPS binding structural motif and the palmitic acid molecule binds in the deep cleft of the predicted substrate binding site suggesting the nature of endogenous acceptor substrate of STF9 resembles palmitic acid. The substrate binding site is covered by a flexible loop which may have involvement in endogenous substrate recognition. Based on the mutational study (Hossain et al., Mol Cell Biochem 350:155-162; 2011) and structural resemblance of STF9-sulfate ion-palmitic acid complex to the hHS3OST3 complex with PAP (3'-phosphoadenosine-5'-phosphate) and an acceptor sugar chain, Glu170 and Arg96 are appeared to be catalytic residues in STF9 sulfuryl transfer mechanism.     

Isotope exchange at equilibrium indicates a steady state ordered kinetic mechanism for human sulfotransferase

Cytosolic sulfotransferase (SULT)-catalyzed sulfation regulates biosignaling molecular biological activities and detoxifies hydroxyl-containing xenobiotics. The universal sulfuryl group donor for SULTcatalyzed sulfation is adenosine 3'-phosphate 5'-phosphosulfate (PAPS). The reaction products are a sulfated product and adenosine 3',5'-diphosphate (PAP). Although the kinetics has been reported since the 1980s,SULT-catalyzed reaction mechanisms remain unclear. Human SULT1A1 catalyzes the sulfation of xenobiotic phenols and has very broad substrate specificity. It has been recognized as one of the most important phase II drug-metabolizing enzymes. Understanding the kinetic mechanism of this isoform is important in understanding drug metabolism and xenobiotic detoxification. In this report, we investigated the SULT1A1-catalyzed phenol sulfation mechanism. The SULT1A1-catalyzed reaction was brought to equilibrium by varying substrate (1-naphthol) and PAPS initial concentrations. Equilibrium constants were determined. Two isotopic exchanges at equilibrium ([14C]1-naphthol <=>[14C]1-naphthyl sulfate and[35S]PAPS<=>[35S]1-naphthyl sulfate) were conducted. First-order kinetics, observed for all the is otopic exchange reactions studied over the entire time scale that was monitored, indicates that the system was truly at equilibrium prior to addition of an isotopic pulse. Complete suppression of the 35S isotopic exchange rate was observed with an increase in the levels of 1-naphthol and 1-naphthyl sulfate in a constant ratio,while no suppression of the 14C exchange rate was observed with an increase in the levels of PAPS and PAP in a constant ratio. Data are consistent with a steady state ordered kinetic mechanism with PAPS and PAP binding to the free enzyme.     

Overexpression of the 3'-phosphoadenosine 5'-phosphosulfate (PAPS) transporter 1 increases sulfation of chondroitin sulfate in the apical pathway of MDCK II cells

The canine 3'-phosphoadenosine 5'-phosphosulfate (PAPS) transporter1 fused to GFP was stably expressed with a typical Golgi localizationin MDCK II cells (MDCK II-PAPST1). The capacity for PAPS uptakeinto Golgi vesicles was enhanced to almost three times thatof Golgi vesicles isolated from untransfected cells. We havepreviously shown that chondroitin sulfate proteoglycans (CSPGs)are several times more intensely sulfated in the basolateralthan the apical secretory pathway in MDCK II cells (Tveit H,Dick G, Skibeli V, Prydz K. 2005. A proteoglycan undergoes differentmodifications en route to the apical and basolateral surfacesof Madin-Darby canine kidney cells. J Biol Chem. 280:29596–29603).Here we demonstrate that increased availability of PAPS in theGolgi lumen enhances the sulfation of CSPG in the apical pathwayseveral times, while sulfation of CSPGs in the basolateral pathwayshows minor changes. Sulfation of heparan sulfate proteoglycansis essentially unchanged. Our data indicate that CSPG sulfationin the apical pathway of MDCK II cells occurs at suboptimalconditions, either because the sulfotransferases involved havehigh K_m values, or there is a lower PAPS concentration in thelumen of the apical secretory route than in the basolateralcounterpart.     

Stereoselective sulfation of terbutaline by the rat liver cytosol: evaluation of experimental approaches

Little is known about the stereochemistry of sulfation of chiral phenolic drugs. In this study we examined several in vitro approaches to this question, using (+)-, (-)-, or (+/-)-terbutaline as the substrate and the rat liver cytosol as the phenolsulfotransferase enzyme source. The cosubstrate PAPS was either generated by the cytosol from inorganic sulfate and ATP or added to the cytosol. The intact sulfate conjugates formed were determined by HPLC. Using the PAPS generating system, which is best suited for the production of relatively large quantities of sulfate conjugates, with the individual enantiomers as substrates, (T)-terbutaline was conjugated to a much greater extent than (-)-terbutaline; the (+)/(-)-enantiomer ratio was 7.3 +/- 0.3 (mean +/- SE). When (+/-)-terbutaline was the substrate and chiral derivatization was employed to separate the sulfate enantiomers formed, a similar (+)/(-)-enantiomer ratio of 7.9 +/- 0.2 was obtained. With PAP35S added to the cytosol, an approach best suited for kinetic studies, the substrate concentration dependence of sulfation could be determined. The Km app for this reaction was identical for (+)- and (-)-terbutaline. However, the Vmax app was 8.1 +/- 0.4 times greater for (+)-terbutaline. This study for the first time shows enantioselectivity in sulfation of a chiral phenolic drug. The experimental approaches used should be valuable for human studies of stereoselective sulfation of terbutaline and other chiral drugs.     

Dermatan is a better substrate for 4-O-sulfation than chondroitin: implications in the generation of 4-O-sulfated, L-iduronate-rich galactosaminoglycans

The biosynthesis of dermatan sulfate is a complex process that involves, inter alia, formation of L-iduronic acid residues by C5-epimerization of D-glucuronic acid residues already incorporated into the growing polymer. It has been shown previously that this reaction is promoted by the presence of the sulfate donor 3'-phosphoadenosine-5'-phosphosulfate. In the present investigation, the role of sulfation in the biosynthesis of L-iduronic acid-rich galactosaminoglycans was examined more closely by a study of the substrate specificities and kinetic properties of the sulfotransferases involved in dermatan sulfate biosynthesis. Comparison of the acceptor reactivities of oligosaccharides from chondroitin and dermatan, in an in vitro system containing microsomes from cultured human skin fibroblasts and 3'-phosphoadenosine-5'-phosphosulfate, showed that Km values for the dermatan fragments were substantially lower than those for their chondroitin counterparts. Calculation of Vmax values likewise showed that dermatan was the better substrate. Whereas dermatan incorporated [35S]sulfate exclusively at the C4 position of N-acetylgalactosamine residues, approximately equal amounts of radioactivity were found at the C4 and C6 positions in the labelled chondroitin. Under standard assay conditions, the 4-O-sulfation of dermatan proceeded about six times faster than the 4-O-sulfation of chondroitin. On the basis of these results, we propose that L-iduronic acids, formed in the course of the biosynthesis of dermatan sulfates, enhance sulfation of their adjacent N-acetylgalactosamine residues, and will thereby be locked in the L-ido configuration.     

Post-translational modification of protein by tyrosine sulfation: active sulfate PAPS is the essential substrate for this modification.

In vitro tyrosine sulfation of recombinant proteins would be a valuable tool in converting those proteins expressed in prokaryotic vectors to their natural form. For this purpose tyrosylprotein sulfotransferase (TPST), the enzyme responsible for tyrosine sulfation of proteins, was characterized from a bovine liver Golgi preparation. TPST was active in a acidic environment with a pH optimum of 6.25, and displayed a stimulation by the Mn2+, with the optimum activity in the presence of 5mM MnCl2. TPST was able to sulfate recombinant hirudin variant 1 (rHV-1) expressed in Escherichia coli and the C-terminal hirudin fragment 54-65 but not the N-terminal hirudin fragment 1-15 by using 3'-phosphoadenosine 5'-phosphosulfate (PAPS), indicating its specificity for the naturally sulfated tyrosine 63. Comparison of the reaction kinetics on synthetic peptides showed that the bovine liver TPST has a higher affinity and reaction rates for those peptides with a aspartyl residue on the N-terminal side of the tyrosine when compared with a glutamyl residue.     

Assay of sulfotransferases.

Two methods are described for the assay of sulfotransferases which are active with sulfate acceptors bearing the hydroxyl functional group. Assays were developed for enzymes which transfer sulfate from 3′-phosphoadenosine–5′-phosphosulfate (PAPS) to sterols, phenols, and simple alcohols thereby forming the corresponding sulfate esters. With a filter binding assay, useful with crude and purified enzyme preparations, a radioactive sterol substrate is used and subsequently separated from labeled product, allowing the determination of between 50 and 400 pmol of product. In a second method, [³⁵S]PAPS is used and the labeled product is separated from PAPS and inorganic sulfate by a thin-layer technique in which product migrates close to the solvent front; the assay is useful with a broad array of substrates and is more sensitive than the filter binding assay.     

Redox control of aryl sulfotransferase specificity

Aryl sulfotransferase IV from rat liver has the very broad substrate range that is characteristic of the enzymes of detoxication. With the conventional assay substrates, 4-nitrophenol and PAPS, sulfation was considered optimal at pH 5.5 whereas the enzyme in the physiological pH range was curiously ineffective. These properties would seem to preclude a physiological function for this cytosolic enzyme. Partial oxidation of the enzyme, however, results not only in a substantial increase in the rate of sulfation of 4-nitrophenol at physiological pH but also in a shift of the pH optimum to this range and radically altered overall substrate specificity. The mechanism for this dependence on redox environment involves oxidation at Cys66, a process previously shown to occur by formation of a mixed disulfide with glutathione or by the formation of an internal disulfide with Cys232. Oxidation at Cys66 acts only as a molecular redox switch and is not directly part of the catalytic mechanism. Underlying the activation process is a change in the nature of the ternary complex formed between enzyme, phenol, and the reaction product, adenosine 3',5'-bisphosphate. The reduced enzyme gives rise to an inhibitory, dead-end ternary complex, the stability of which is dictated by the ionization of the specific phenol substrate. Ternary complex formation impedes the binding of PAPS that is necessary to initiate a further round of the reaction and is manifest as profound, substrate-dependent inhibition. In contrast, the ternary complex formed when the enzyme is in the partially oxidized state allows binding of PAPS and the unhindered completion of the reaction cycle.     

Defect in 3′-phosphoadenosine 5′-phosphosulfate synthesis in brachymorphic mice: I. Characterization of the defect

Biosynthesis of the undersulfated proteoglycan found in brachymorphic mouse (bm/ bm) cartilage has been investigated. Similar amounts of cartilage proteoglycan core protein, as measured by radioimmune inhibition assay, and comparable activity levels of four of the glycosyltransferases requisite for synthesis of chondroitin sulfate chains were found in cartilage homogenates from neonatal bm/bm and normal mice, suggesting normal production of glycosylated core protein acceptor for sulfation. When incubated with ³⁵S-labeled 3′-phosphoadenosine 5′-phosphosulfate (PAPS), bm/bm cartilage extracts showed a higher than control level of sulfotransferase activity. In contrast, when synthesis was initiated from ATP and ³⁵SO₄^2?, mutant cartilage extracts showed lower incorporation of ³⁵SO₄^2? into endogenous chondroitin sulfate proteoglycan (19% of control level) and greatly reduced formation of PAPS (10% of control level). Results from coincubations of normal and mutant cartilage extracts exhibited intermediate levels of sulfate incorporation into PAPS and endogenous acceptors, suggesting the absence of an inhibitor for sulfate-activating enzymes or sulfotransferases. Degradation rates of ³⁵S]PAPS and of ³⁵S-labeled adenosine 5′-phosphosulfate (APS) were comparable in bm/bm and normal cartilage extracts. Specific assays for both ATP sulfurylase (sulfate adenylyltransferase; ATP:sulfate adenylyltransferase, EC 2.7.7.4) and APS kinase (adenylylsulfate kinase; ATP:adenylylsulfate 3′-phosphotransferase, EC 2.7.1.25) showed decreases in the former (50% of control) and the latter (10–15% of control) enzyme activities in bm/bm cartilage extracts. Both enzyme activities were reduced to intermediate levels in extracts of cartilage from heterozygous brachymorphic mice (ATP-sulfurylase, 80% of control; APS kinase, 40–70% of control). Furthermore, the moderate reduction in ATP sulfurylase activity in bm/bm cartilage extracts was accompanied by increased lability to freezing and thawing of the residual activity of this enzyme. These results indicate that under-sulfation of chondroitin sulfate proteoglycan in bm/bm cartilage is due to a defect in synthesis of the sulfate donor (PAPS), resulting from diminished activities of both ATP sulfurylase and APS kinase, although the reduced activity of the latter enzyme seems to be primarily responsible for the defect in PAPS synthesis.     

5'-adenosinephosphosulfate lies at a metabolic branch point in mycobacteria

Bacterial sulfate assimilation pathways provide for activation of inorganic sulfur for the biosynthesis of cysteine and methionine, through either adenosine 5'-phosphosulfate (APS) or 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as intermediates. PAPS is also the substrate for sulfotransferases that produce sulfolipids, putative virulence factors, in Mycobacterium tuberculosis such as SL-1. In this report, genetic complementation using Escherichia coli mutant strains deficient in APS kinase and PAPS reductase was used to define the M. tuberculosis and Mycobacterium smegmatis CysH enzymes as APS reductases. Consequently, the sulfate assimilation pathway of M. tuberculosis proceeds from sulfate through APS, which is acted on by APS reductase in the first committed step toward cysteine and methionine. Thus, M. tuberculosis most likely produces PAPS for the sole use of this organism's sulfotransferases. Deletion of CysH from M. smegmatis afforded a cysteine and methionine auxotroph consistent with a metabolic branch point centered on APS. In addition, we have redefined the substrate specificity of the B. subtilis CysH, formerly designated a PAPS reductase, as an APS reductase, based on its ability to complement a mutant E. coli strain deficient in APS kinase. Together, these studies show that two conserved sequence motifs, CCXXRKXXPL and SXGCXXCT, found in the C termini of all APS reductases, but not in PAPS reductases, may be used to predict the substrate specificity of these enzymes. A functional domain of the M. tuberculosis CysC protein was cloned and expressed in E. coli, confirming the ability of this organism to make PAPS. The expression of recombinant M. tuberculosis APS kinase provides a means for the discovery of inhibitors of this enzyme and thus of the biosynthesis of SL-1.     

Reaction product affinity regulates activation of human sulfotransferase 1A1 PAP sulfation

Cytosolic sulfotransferase (SULT)-catalyzed sulfation regulates the activity of bio-signaling molecules and aids in metabolizing hydroxyl-containing xenobiotics. The sulfuryl donor for the SULT reaction is adenosine 3′-phosphate 5′-phosphosulfate (PAPS), while products are adenosine 3′,5′-diphosphate (PAP) and a sulfated alcohol. Human phenol sulfotransferase (SULT1A1) is one of the major detoxifying enzymes for phenolic xenobiotics. The mechanism of SULT1A1-catalyzed sulfation of PAP by pNPS was investigated. PAP was sulfated by para-nitrophenyl sulfate (pNPS) in a concentration-dependent manner. 2-Naphthol inhibited sulfation of PAP, competing with pNPS, while phenol activated the sulfation reaction. At saturating PAP, a ping pong kinetic mechanism is observed with pNPS and phenol as substrates, consistent with phenol intercepting the E–PAPS complex prior to dissociation of PAPS. At high concentrations, phenol competes with pNPS, consistent with formation of the E–PAP–phenol dead-end complex. Data are consistent with the previously reported mechanism for sulfation of 2-naphthol by PAPS, and its activation by pNPS [14]. Overall, data are consistent with release of PAP from E–PAP and PAPS from E–PAPS contributing to rate-limitation in both reaction directions.     

The effect of chondroitin sulfate molecular weight and degree of sulfation on the activity of a sulfotransferase from chicken embryo epiphyseal cartilages

The transfer of [35S] sulfate from [35S]PAPS, by means of PAPS: chondroitin sulfate sulfotransferase, to various chondroitin sulfates, with different degrees of sulfation and molecular weights is reported. Analyses by digestion with chondroitin AC and specific 4- or 6-sulfatases indicate that the sulfation occurs only in position 6 of the non-sulfated N-acetyl galactosamine moiety. The 50-70% desulfated chondroitin 4/6-sulfates are two times better sulfate acceptors than totally desulfated chondroitin, and the affinity of the sulfotransferase increases markedly from the octa-to the deca-saccharide. These results suggest that sulfation increases sharply only after the growing polysaccharide contains about 10 sugar residues, in the early stages of polymerization, and that the sulfation of chondroitin sulfate may be a process in which the addition of some sulfate groups facilitates further sulfation.