Crystal Structure of the Human Estrogen Sulfotransferase-PAPS Complex

Abstract

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 17β-estradiol, the ternary structure with the PAPS and estradiol molecule, is modeled. These structures have now provided a more complete view of the SN2-like in-line displacement reaction catalyzed by sulfotransferases. In the PAPS-bound structure, the side chain nitrogen of the catalytic Lys47 interacts with the side chain hydroxyl of Ser137 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 Lys47 with Ser137 may regulate PAPS hydrolysis in the absences of an acceptor substrate. Supporting the structural data, the mutations of Ser137 to cysteine and alanine decrease gradually kcat for PAPS hydrolysis and transfer activity. Thus, Ser137 appears to play an important role in regulating the side chain interaction of Lys47 with the bridging oxygen between the 5′-phosphate and the sulfate of PAPS. 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 17β-estradiol, the ternary structure with the PAPS and estradiol molecule, is modeled. These structures have now provided a more complete view of the SN2-like in-line displacement reaction catalyzed by sulfotransferases. In the PAPS-bound structure, the side chain nitrogen of the catalytic Lys47 interacts with the side chain hydroxyl of Ser137 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 Lys47 with Ser137 may regulate PAPS hydrolysis in the absences of an acceptor substrate. Supporting the structural data, the mutations of Ser137 to cysteine and alanine decrease gradually kcat for PAPS hydrolysis and transfer activity. Thus, Ser137 appears to play an important role in regulating the side chain interaction of Lys47 with the bridging oxygen between the 5′-phosphate and the sulfate of PAPS. Sulfuryl transfer, also referred to as sulfation or sulfonation, is the transfer reaction of the sulfate group from the ubiquitous donor 3′-phosphoadenosine 5′-phosphosulfate (PAPS) 1The abbreviations used are: PAPS3′-phosphoadenosine 5′-phosphosulfateMES2-(N-morpholino)ethanesulfonic acidPAP3′-phosphoadenosine 5′-phosphateE217β-estradiolhESThuman estrogen sulfotransferasemESTmouse estrogen sulfotransferase to an acceptor substrate (1Leyh T.S. Crit. Rev. Biochem. Mol. Biol. 1993; 28: 515-542Crossref PubMed Scopus (91) Google Scholar, 2Duffel M.W. Guegenerich F.P. Comprehensive Toxicology. 3. Oxford University Press, Oxford, United Kingdom1997: 365-386Google Scholar, 3Falany C.N. FASEB J. 1997; 11: 1-2Crossref PubMed Scopus (68) Google Scholar). A large family of enzymes known as sulfotransferases catalyzes these reactions. The active sites of sulfotransferase enzymes are conserved in all of the crystal structures solved for sulfotransferases (4Kakuta Y. Pedersen L.G. Carter C.W. Negishi M. Pedersen L.C. Nat. Struct. Biol. 1997; 4: 904-908Crossref PubMed Scopus (233) Google Scholar, 5Kakuta Y. Petrotchenko E. Pedersen L.C. Negishi M. J. Biol. Chem. 1998; 273: 27325-27330Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 6Kakuta Y. Pedersen L.G. Pedersen L.C. Negishi M. Trends Biochem. Sci. 1998; 23: 129-130Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 7Kakuta Y. Sueyoshi T. Negishi M. Pedersen L.C. J. Biol. Chem. 1999; 274: 10673-10676Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 8Dajani R. Cleasby A. Neu M. Wonacott A.J. Jhoti H. Hood A.M. Modi S. Hersey A. Taskinen J. Cooke R.M. Manchee G.R. Coughtrie M.W.H. J. Biol. Chem. 1999; 274: 37862-37868Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 9Bidwell L.M. McManus M.E. Gaedigk A. Kakuta Y. Negishi M. Pedersen L. Martin J.F. J. Mol. Biol. 1999; 293: 521-530Crossref PubMed Scopus (99) Google Scholar, 10Pedersen L.C. Petrotchenko E.V. Negishi M. FEBS Lett. 2000; 475: 61-64Crossref PubMed Scopus (91) Google Scholar). In the mEST structure with both PAP and E2, conserved residues Ser138 and Lys48 directly interact with the 3′- and 5′-phosphate of the PAP molecule, respectively, whereas the conserved His108 directly coordinates to the acceptor group of the 17β-estradiol (E2) molecule, suggesting it to be the catalytic base. The crystal structure in the presence of PAP and orthovanadate mimics the transition state of the transfer reaction in which the lysine residue directly coordinates to the bridging oxygen of the 5′-phosphate of the PAP (5Kakuta Y. Petrotchenko E. Pedersen L.C. Negishi M. J. Biol. Chem. 1998; 273: 27325-27330Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). This coordination suggested that the lysine could be a catalytic residue participating in the dissociation of the sulfate group from PAPS. Site-directed mutagenesis studies have confirmed the functional importance of this residue in various sulfotransferase enzymes (5Kakuta Y. Petrotchenko E. Pedersen L.C. Negishi M. J. Biol. Chem. 1998; 273: 27325-27330Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar,11Marsolais F. Varin L. J. Biol. Chem. 1995; 270: 30458-30463Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 12Sueyoshi T. Kakuta Y. Pedersen L.C. Wall F.E. Pedersen L.G. Negishi M. FEBS Lett. 1998; 433: 211-214Crossref PubMed Scopus (47) Google Scholar, 13Ong E. Yeh J.-C. Ding Y. Hindsgaul O. Pedersen L.C. Negishi M. Fukuda M. J. Biol. Chem. 1999; 274: 25608-25612Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Accordingly, the transfer reaction has been proposed to proceed through an SN2-like in-line displacement mechanism in which the conserved lysine and histidine play essential roles. 3′-phosphoadenosine 5′-phosphosulfate 2-(N-morpholino)ethanesulfonic acid 3′-phosphoadenosine 5′-phosphate 17β-estradiol human estrogen sulfotransferase mouse estrogen sulfotransferase The serine residue is conserved in all known sulfotransferases with no exception. Site-directed mutagenesis of the conserved serine has been shown to abrogate the activity of human natural killer cell-1 sulfotransferase (13Ong E. Yeh J.-C. Ding Y. Hindsgaul O. Pedersen L.C. Negishi M. Fukuda M. J. Biol. Chem. 1999; 274: 25608-25612Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Despite the apparent functional importance, the structural implication of the conserved serine is not clear at this time. In all known crystal structures, the conserved serine forms a hydrogen bond with the 3′-phosphate of the PAP molecule and is away from the 5′-phosphate group where the transfer occurs. Previous structures provided no evidence for the involvement of the serine residue in catalysis, although the interaction with the 3′-phosphate group suggested that it might be conserved for the specific binding of PAPS. The lack of a sulfotransferase structure with the active donor PAPS has limited our understanding of the reaction mechanism and the roles conserved residues like the serine might play in catalysis. We have now crystallized the V269E mutant (a surface mutation) of hEST in the presence of PAPS. This hEST mutant converts the enzyme from a dimer to a monomer in solution but has no observable effect on the activity (14Petrotchenko E.V. Pedersen L.C. Brochers C.H. Tomer K.B. Negishi M. FEBS Lett. 2001; 490: 39-43Crossref PubMed Scopus (90) Google Scholar). All residues involved in interacting directly with the donor and acceptor substrates are found to be conserved in the hEST as observed in the crystal structures of mEST (4Kakuta Y. Pedersen L.G. Carter C.W. Negishi M. Pedersen L.C. Nat. Struct. Biol. 1997; 4: 904-908Crossref PubMed Scopus (233) Google Scholar). However in the hEST-PAPS structure, a conformational change in the position of Lys47is observed. In this structure, Lys47 forms a hydrogen bond with Ser137 instead of with the bridging oxygen between the 5′-phosphate and the sulfate group of PAPS. Complemented with site-directed mutagenesis studies on Ser137 and His107, we herein present experimental evidence that leads us to propose the reaction mechanism in which the 3′-phosphate-Ser interaction helps regulates the action of the lysine in controlling the dissociation of the 5′-sulfate group from PAPS in the absence of substrate. Upon binding of substrate and initiation of catalysis by His107, Lys47 undergoes a conformational change and interacts with the bridging oxygen of the phosphate-sulfate bond of PAPS to promote the dissociation of the PAP leaving group. A hEST cDNA, kindly provided by Charles Falany, was cloned into pGEX-4T3 plasmid. Site-directed mutagenesis was performed using QuikChange kit (Stratagene) and verified by DNA sequencing. Each recombinant plasmid was transformed into Escherichia coli BL21DE3 cell, and the expressed recombinant enzyme was bound on glutathione-Sepharose from which the pure enzyme was eluted by thrombin digestion. For crystallization, the purified enzyme was then washed over a bensoyl-Sepharose microcolumn followed by extensive dialysis against 0.5 mmNaH2PO4 and 100 mm NaCl at pH 7.2. The crystals of hEST were obtained using the sitting drop vapor diffusion technique. Protein concentrated at 15 mg/ml in 0.5 mmNaH2PO4, 100 mm NaCl, and 4 mm PAP (Sigma), pH 7.2, was mixed in equal volume with 0.1m MES, pH 6.0, and 18% polyethylene glycol 8000 and then placed at 20 °C. Typical crystals appeared after 10 days and grew to 0.5 × 0.5 × 0.02 mm after one month. For data collection, crystals were transferred into the cold room at 4 °C. After the temperature in the drop equilibrated, the crystals were transferred three times into 60 μl of 0.1 m MES, pH 6.0, 22% polyethylene glycol 8000, and 10 mm PAPS and allowed to sit for 4 h. Crystals were then transferred in four steps of increasing ethylene glycol concentration until a final concentration of 15% ethylene glycol in the soaking solution was obtained. Crystals were frozen in the nitrogen stream at −180 °C. Data were collected on a RaxisIV image plate detector with a RU3H-rotating anode generator. All data were processed using Denzo and Scalepack (15Otwinowski Z. Minor W. Methods Enzymol. 1996; 276: 307-326Crossref Scopus (38617) Google Scholar). The structure factor phases were determined by molecular replacement using the program AmoRe (16Navaza J. Acta Crystallogr. Sec. A. 1994; 50: 157-168Crossref Scopus (5030) Google Scholar). The hEST crystals obtained were from the mutant V269E. A monomer of mEST coordinates with a PDB identification of 1AQU was used as the initial search model against data from the V269E mutant in complex with PAP crystal. The V269E structure was refined with multiple cycles of model building using the program “O” (17Jones T.A. Zou J.Y. Cowan S.W. Kjeledgaard M. Acta Crystallogr. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar) for model building and CNS (18Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Acta Crystallogr. Sec. D. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar) for refinement. These coordinates were then used as a starting model for the structures reported here, which were refined and built in a similar fashion (Table I). The quality of the models was checked using Procheck (19Collaborative Computational Project Number 4Acta Crystallogr. Sec. D. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar). The position of Lys47 was confirmed using Fo− Fc, 2 Fo −Fc, and Fo −Fc difference maps as well as omit maps. In addition, the Lys residue was modeled and refined in both orientations to determine the correct side chain orientation. The PDB identifier code is 1HY3 for these coordinates.Table ICrystallographic data statisticsData setPAP + estradiolPAPSUnit cell dimensionsa = 61.64, b = 96.65, c = 62.79a = 61.93, b = 97.26, c = 62.73α = 90 °, β = 92.70 °, γ = 90 °α = 90 °, β = 91.13 °, γ = 90 °Space groupP21P21No. of observations81,110182,966Unique reflections25,47263,064Rsym (%) (last shell)1-aRsym = ΣhΣi‖Ii(h) − <I(h)>‖/ΣhΣiIi(h).7.8 (33.0)4.9 (22.9)I/ςI (last shell)12.5 (2.7)12.87 (2.2)Mosaicity0.680.62Completeness (%) (last shell)98.3 (86.8)91.8 (52.1)Refinement statisticsResolution (Å)50–2.550–1.8Rcryst(%)1-aRsym = ΣhΣi‖Ii(h) − <I(h)>‖/ΣhΣiIi(h).18.7019.7Rfree22.521.7No. of waters2684501. Root mean square deviation from ideal values Bond length (Å)0.0080.006 Bond angle (°)1.21.3 Dihedral angle (°)21.421.4 Improper angle (°)0.820.72 Mean B value (Å2)27.927.32. Ramachandran statisticsResidues in: most favored regions (%)91.192.2 additionally allowed regions8.77.4 generously allowed regions0.20.0 disallowed regions0.00.2b Rcryst = Σ‖‖Fo‖ − ‖Fc‖/Σ‖Fo‖ calculated from working data set. Rfree is calculated from 5% of data randomly chosen not to be included in refinement.1-a Rsym = ΣhΣi‖Ii(h) − <I(h)>‖/ΣhΣiIi(h). Open table in a new b Rcryst = Σ‖‖Fo‖ − ‖Fc‖/Σ‖Fo‖ calculated from working data set. Rfree is calculated from 5% of data randomly chosen not to be included in refinement. PAPS was purified through using 20 mm pH and of NaCl A Y. Pedersen L.C. D. R. Carter C.W. Negishi M. Biochem. 1998; PubMed Scopus Google Scholar) was used to estrogen sulfotransferase activity and to of PAPS and PAPS the containing enzyme and pure PAPS in 20 mm pH was for 15 at °C. μl of of the reaction was on using the of and were used for the of PAPS and The crystal structures of hEST have now been solved in the presence of PAPS and of PAP and The structure of hEST is a The of the molecule is of an This a β of by on both and a conserved the of the This of the molecule the conserved and that the binding for the donor substrate PAPS A the in the the from the containing residues in These the interaction as seen in human sulfotransferase and human sulfotransferase crystals that has been to be the (14Petrotchenko E.V. Pedersen L.C. Brochers C.H. Tomer K.B. Negishi M. FEBS Lett. 2001; 490: 39-43Crossref PubMed Scopus (90) Google Scholar). the V269E not the hEST from the dimer in the crystal The of the acceptor of hEST is similar to that of mEST (4Kakuta Y. Pedersen L.G. Carter C.W. Negishi M. Pedersen L.C. Nat. Struct. Biol. 1997; 4: 904-908Crossref PubMed Scopus (233) Google Scholar). These the molecule in the orientation. The side from residues and hydrogen with the acceptor hydroxyl of the molecule as is also seen in the mEST structures (4Kakuta Y. Pedersen L.G. Carter C.W. Negishi M. Pedersen L.C. Nat. Struct. Biol. 1997; 4: 904-908Crossref PubMed Scopus (233) Google Scholar). The in the the group of the This is by the acid from mEST to to to to to to to and to this of the hEST structure, a of from to appears to have a from that of mEST and appears to have on the substrate. The PAP of PAPS molecule in the hEST-PAPS structure well with the PAP in the The hEST-PAPS crystal structure the catalytic residues that with the sulfate The of the sulfate group is in position to a hydrogen bond with of and the from Lys47 The sulfate coordination to these residues is similar to that of the molecule as observed in the structure of the (5Kakuta Y. Petrotchenko E. Pedersen L.C. Negishi M. J. Biol. Chem. 1998; 273: 27325-27330Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). The is from the acceptor group of the molecule the hEST-PAPS structure is with the structure The position of the sulfate group with to the acceptor substrate molecule in this is with the proposed in-line transfer reaction mechanism on the of the hEST-PAPS structure with the The hydrogen in the PAPS bound structure are by whereas in the structure are with J. Crystallogr. 1991; Google Scholar) and Methods Enzymol. 1997; PubMed Scopus Google Scholar) were used to this image The most structural difference in the which from the is the side chain of The of Lys47 interacts with the leaving oxygen of 5′-phosphate group in the PAP bound structures as observed in the structures (4Kakuta Y. Pedersen L.G. Carter C.W. Negishi M. Pedersen L.C. Nat. Struct. Biol. 1997; 4: 904-908Crossref PubMed Scopus (233) Google Scholar, 7Kakuta Y. Sueyoshi T. Negishi M. Pedersen L.C. J. Biol. Chem. 1999; 274: 10673-10676Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 8Dajani R. Cleasby A. Neu M. Wonacott A.J. Jhoti H. Hood A.M. Modi S. Hersey A. Taskinen J. Cooke R.M. Manchee G.R. Coughtrie M.W.H. J. Biol. Chem. 1999; 274: 37862-37868Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 10Pedersen L.C. Petrotchenko E.V. Negishi M. FEBS Lett. 2000; 475: 61-64Crossref PubMed Scopus (91) Google Scholar). The angle of the lysine has in the hEST-PAPS structure with in the PAP-bound a the of Lys47 is found to to the side chain oxygen of Ser137 in the PAPS bound structure and not to the bridging oxygen between the 5′-phosphate and sulfate groups of the PAPS In addition, the position of has also been by the position of A Fo −Fc omit the position of the lysine side chain in the PAPS structure A Fo −Fc difference was also calculated for the PAPS structure with Lys47 modeled and refined in the bound The at the position of the as well as the position of the evidence that the position of the lysine side chain in the PAPS-bound structure is modeled The difference in the of Lys47 may be of a difference on the bridging oxygen between the 5′-phosphate and sulfate The on the 5′-phosphate of PAP is the on the in PAPS is In the PAP-bound structure, is a at the position of the bridging oxygen the oxygen is not binding a group. is no on the bridging oxygen in the PAPS molecule, the of with the of Ser137 that also forms a hydrogen bond with the 3′-phosphate of the PAPS The interaction of Ser137 with the catalytic residue Lys47 a catalytic role for Ser137 as The conserved lysine Lys47 in is as a catalytic residue that in the dissociation of the sulfate group from PAPS the sulfotransferase reaction (4Kakuta Y. Pedersen L.G. Carter C.W. Negishi M. Pedersen L.C. Nat. Struct. Biol. 1997; 4: 904-908Crossref PubMed Scopus (233) Google Scholar, 5Kakuta Y. Petrotchenko E. Pedersen L.C. Negishi M. J. Biol. Chem. 1998; 273: 27325-27330Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar,11Marsolais F. Varin L. J. Biol. Chem. 1995; 270: 30458-30463Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, F. Varin L. J. Biochem. 1997; PubMed Scopus Google Scholar). For we that the of the Lys48 to estrogen sulfotransferase activity of the whereas the mutant a of the activity (5Kakuta Y. Petrotchenko E. Pedersen L.C. Negishi M. J. Biol. Chem. 1998; 273: 27325-27330Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). the crystal structure of the complex that the state of the transfer these site-directed mutagenesis studies that the role of the conserved lysine in catalysis is to as a donor to the leaving PAP group (5Kakuta Y. Petrotchenko E. Pedersen L.C. Negishi M. J. Biol. Chem. 1998; 273: 27325-27330Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). In of the that the side chain nitrogen of Lys47 coordinates to Ser137 and not to the bridging oxygen in the PAPS-bound structure, we that the side chain interaction of Lys47 with Ser137 may regulate the catalytic activity of this Ser137 of hEST was to alanine or and the enzymes were for the PAPS hydrolysis the of PAPS for estrogen sulfotransferase activity was for the to the PAPS concentration used for the of PAPS hydrolysis of the and enzymes was in the presence of PAPS concentration of PAPS PAPS concentration the effect mutations have on the of catalysis change in the binding for PAPS. The hEST of PAPS values (Table This suggested that that Ser137 was for the binding of PAPS to the which was with binding to the 3′-phosphate group of the PAPS The role of Ser137 in regulating the catalytic of Lys47 was with of PAPS. on the kcat of the PAPS hydrolysis with the whereas the mutant hydrolysis (Table Thus, the of Ser137 may be to hydrolysis in the absence of the substrate. with the kcat of hydrolysis the mutations also kcat for estrogen sulfotransferase activity of the These that Ser137 is of regulating the hydrolysis as well as the sulfation of the substrate. Ser137 not directly interact with the bridging oxygen between the 5′-phosphate and sulfate groups of the PAPS molecule, Ser137 may regulate the dissociation through side chain interaction with or the interaction with Ser137 could the side chain of Lys47 to a more position to the bridging oxygen in the of These mutations that this which is not found the 5′-phosphate or sulfate regulate the hydrolysis and sulfotransferase activity of the these studies into the crystal structures are with a reaction this is the of Ser137 with of the and hEST of of sulfotransferase activity was as and a reported was used to the of PAPS and kcat of sulfation values Y. Pedersen L.C. D. R. Carter C.W. Negishi M. Biochem. 1998; PubMed Scopus Google Scholar). For kcat of the activity of PAPS hydrolysis was using of enzyme with and PAPS. The kcat of hydrolysis values are the of at for not Open table in a new The sulfotransferase activity was as and a reported was used to the of PAPS and kcat of sulfation values Y. Pedersen L.C. D. R. Carter C.W. Negishi M. Biochem. 1998; PubMed Scopus Google Scholar). For kcat of the activity of PAPS hydrolysis was using of enzyme with and PAPS. The kcat of hydrolysis values are the of at for not For the transfer reaction to the side chain of Lys47 from interacting with Ser137 to interacting with the bridging oxygen catalysis. The as to this conformational is that the binding of could a structural that the side chain position of no structural change is observed binding is that an change in the of the lysine could the In the transfer the catalytic histidine the acceptor of the molecule and the of this hydroxyl group. the acceptor group the which a on the bridging oxygen of the PAPS This may be to the of the lysine from interacting with the serine to interacting with the this was to to histidine the and the of the mutant were alanine that were (5Kakuta Y. Petrotchenko E. Pedersen L.C. Negishi M. J. Biol. Chem. 1998; 273: 27325-27330Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, F. Varin L. J. Biochem. 1997; PubMed Scopus Google Scholar, Y. Y. M. M. 1996; PubMed Scopus Google the mutant was expressed as a to as a as the enzyme in E. coli The mutant was to enzyme for estrogen sulfotransferase and PAPS hydrolysis activity was observed with the mutant for both sulfotransferase and the of for catalysis (Table These that the action of the catalytic histidine is essential for the side chain nitrogen of Lys47 to from Ser137 to the bridging oxygen and to the catalysis. all structural and the proposed in-line displacement reaction mechanism is in The conserved serine an important role in controlling PAPS hydrolysis activity of sulfotransferase. The binding of PAPS to the enzyme Ser137 to an interaction with This interaction the side chain nitrogen of Lys47 from the bridging PAPS Upon binding of the group a that the building a on the bridging The then the side chain of Lys47 to to the bridging in sulfate dissociation and transfer by the transition state and the to the bridging Thus, a of this reaction be as on a coordination chain starting from the 3′-phosphate of PAPS molecule to the bridging oxygen between the 5′-phosphate and sulfate group. The and to in to regulate the the transfer We Charles of at for a hEST We Pedersen of at and for