The Sulfuryl Transfer Mechanism

Estrogen sulfotransferase (EST) catalyzes transfer of the 5′-sulfuryl group of adenosine 3′-phosphate 5′-phosphosulfate (PAPS) to the 3α-phenol group of estrogenic steroids such as estradiol (E2). The recent crystal structure of EST-adenosine 3′,5′-diphosphate (PAP)- E2complex has revealed that residues Lys48, Thr45, Thr51, Thr52, Lys106, His108, and Try240 are in position to play a catalytic role in the sulfuryl transfer reaction of EST (Kakuta Y., Pedersen, L. G., Carter, C. W., Negishi, M., and Pedersen, L. C. (1997) Nat. Struct. Biol. 4, 904–908). Mutation of Lys48, Lys106, or His108 nearly abolishes EST activity, indicating that they play a critical role in catalysis. A present 2.2-Å resolution structure of EST-PAP-vanadate complex indicates that the vanadate molecule adopts a trigonal bipyramidal geometry with its equatorial oxygens coordinated to these three residues. The apical positions of the vanadate molecule are occupied by a terminal oxygen of the 5′-phosphate of PAP (2.1 Å) and a possible water molecule (2.3 Å). This water molecule superimposes well to the 3α-phenol group of E2 in the crystal structure of the EST·PAP·E2 complex. These structures are characteristic of the transition state for an in-line sulfuryl transfer reaction from PAPS to E2. Moreover, residues Lys48, Lys106, and His108 are found to be coordinated with the vanadate molecule at the transition state of EST. Estrogen sulfotransferase (EST) catalyzes transfer of the 5′-sulfuryl group of adenosine 3′-phosphate 5′-phosphosulfate (PAPS) to the 3α-phenol group of estrogenic steroids such as estradiol (E2). The recent crystal structure of EST-adenosine 3′,5′-diphosphate (PAP)- E2complex has revealed that residues Lys48, Thr45, Thr51, Thr52, Lys106, His108, and Try240 are in position to play a catalytic role in the sulfuryl transfer reaction of EST (Kakuta Y., Pedersen, L. G., Carter, C. W., Negishi, M., and Pedersen, L. C. (1997) Nat. Struct. Biol. 4, 904–908). Mutation of Lys48, Lys106, or His108 nearly abolishes EST activity, indicating that they play a critical role in catalysis. A present 2.2-Å resolution structure of EST-PAP-vanadate complex indicates that the vanadate molecule adopts a trigonal bipyramidal geometry with its equatorial oxygens coordinated to these three residues. The apical positions of the vanadate molecule are occupied by a terminal oxygen of the 5′-phosphate of PAP (2.1 Å) and a possible water molecule (2.3 Å). This water molecule superimposes well to the 3α-phenol group of E2 in the crystal structure of the EST·PAP·E2 complex. These structures are characteristic of the transition state for an in-line sulfuryl transfer reaction from PAPS to E2. Moreover, residues Lys48, Lys106, and His108 are found to be coordinated with the vanadate molecule at the transition state of EST. adenosine 3′-phosphate 5′-phosphosulfate adenosine 3′,5′-diphosphate estradiol estrogen sulfotransferase. Sulfuryl transfer reactions are widely observed in various biological processes and are conserved from bacterium to human. As a result, deficiencies due to the lack of metabolic activation of sulfate can be lethal in humans (1Superti-Furga A. Am. J. Hum. Genet. 1994; 55: 1137-1145PubMed Google Scholar). Various biological signaling molecules including neurotransmitters (2Roth J.A. Trends Biochem. Sci. 1986; 11: 404-407Abstract Full Text PDF Scopus (0) Google Scholar), steroid hormones (3Strott C.A. Endocr. Rev. 1996; 17: 670-697Crossref PubMed Scopus (166) Google Scholar, 4Hobkirk R. Trends Endocrinol. Metab. 1993; 4: 69-74Abstract Full Text PDF PubMed Scopus (119) Google Scholar), and peptides and proteins (5Matsubayashi Y. Takagi L. Sakagami Y. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13357-13362Crossref PubMed Scopus (114) Google Scholar, 6Ouyang Y. Lane W.S. Moore K., L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2896-2901Crossref PubMed Scopus (144) Google Scholar) can be sulfated to alter biological activity. Growth factors and blood coagulation factors become fully activated after binding to glucosaminoglycan sulfates such as heparan sulfates (7Rosenberg R.D. Shworak N.W. Liu J. Schwartz J.J. Zhang L. J. Clin. Invest. 1997; 99: 2062-2070Crossref PubMed Scopus (254) Google Scholar, 8Nelson R.M. Venot A. Bevilacqua M.P. Linhardt R.J. Stamenkovic I. Annu. Rev. Cell Dev. Biol. 1995; 11: 601-631Crossref PubMed Scopus (101) Google Scholar). The sulfation/desulfation balance, thus, regulates cell growth and differentiation as well as neuronal and hormonal homeostasis. Sulfation generally detoxifies xenobiotics such as pharmaceutical drugs and synthetic and naturally occurring chemicals (9Falany C.N. FASEB J. 1997; 11: 206-216Crossref PubMed Scopus (517) Google Scholar). In some cases, however, xenobitic sulfation can be used for the in vivoactivation of prodrugs or can result in potentiating toxicity and carcinogenicity (10Meisheri K. Cipkus L.A. Taylor C.J. J. Pharmacol. Exp. Ther. 1987; 245: 751-760Google Scholar, 11Glatt H. FASEB J. 1997; 11: 314-321Crossref PubMed Scopus (184) Google Scholar). Sulfation of these diverse substrates is catalyzed by a group of enzymes referred to as sulfotransferases that use adenosine 3′-phosphate 5′-phosphosulfate (PAPS)1 as the ubiquitous sulfate donor.The crystal structure of the mouse estrogen sulfotransferase (EST) was the first three-dimensional structure to be solved for the sulfotransferase family (12Kakuta Y. Pedersen L.C. Chae K. Song W.-C. Leblanc D. London R. Cater C.W. Negishi M. Biochem. Pharmacol. 1998; 55: 313-317Crossref PubMed Scopus (27) Google Scholar, 13Kakuta Y. Pedersen L.G. Carter C.W. Negishi M. Pedersen L.C. Nat. Struct. Biol. 1997; 4: 904-908Crossref PubMed Scopus (231) Google Scholar). The structure revealed the catalytic core of the enzyme to be remarkably similar to those of nucleotide kinases, despite its little to no sequence homology to the kinases. The 5′-phosphate of the inactive cofactor adenosine 3′,5′-diphosphate (PAP) and the 3α-phenol group of estradiol (E2) in the EST·PAP·E2 structure superimpose well to the leaving β-phosphate group of ADP and the entering phosphate group of AMP in the UK-ADP-AMP structure, respectively (13Kakuta Y. Pedersen L.G. Carter C.W. Negishi M. Pedersen L.C. Nat. Struct. Biol. 1997; 4: 904-908Crossref PubMed Scopus (231) Google Scholar, 14Nuller-Dieckmann H. Schulz G.E.J. J. Mol. Biol. 1994; 236: 361-367Crossref PubMed Scopus (80) Google Scholar). These structural relationships, as well as an earlier study with phenol sulfotransferase (15Duffel M.W. Jakoby W.B. J. Biol. Chem. 1981; 256: 11123-11127Abstract Full Text PDF PubMed Google Scholar), suggest that the catalytic mechanism of sulfuryl and phosphoryl transfers may be similar. The lack of transition state information on the EST structure has made it difficult to visualize the sulfuryl transfer mechanism. The EST·PAP·E2 structure previously identified Lys48, Thr45, Thr51, The52, Lys106, His108, and Tyr240 as possible catalytic residues. We have performed site-directed mutagenesis studies on these residues in order to better understand the sulfuryl transfer mechanism of EST, and the roles of these residues in catalysis. Moreover, vanadate has been employed in crystallographic analysis in order to obtain a model of the transition state of EST. Based on these studies, we have identified residues involved in stabilizing the transition state that promotes the sulfuryl transfer reaction. Sulfuryl transfer reactions are widely observed in various biological processes and are conserved from bacterium to human. As a result, deficiencies due to the lack of metabolic activation of sulfate can be lethal in humans (1Superti-Furga A. Am. J. Hum. Genet. 1994; 55: 1137-1145PubMed Google Scholar). Various biological signaling molecules including neurotransmitters (2Roth J.A. Trends Biochem. Sci. 1986; 11: 404-407Abstract Full Text PDF Scopus (0) Google Scholar), steroid hormones (3Strott C.A. Endocr. Rev. 1996; 17: 670-697Crossref PubMed Scopus (166) Google Scholar, 4Hobkirk R. Trends Endocrinol. Metab. 1993; 4: 69-74Abstract Full Text PDF PubMed Scopus (119) Google Scholar), and peptides and proteins (5Matsubayashi Y. Takagi L. Sakagami Y. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13357-13362Crossref PubMed Scopus (114) Google Scholar, 6Ouyang Y. Lane W.S. Moore K., L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2896-2901Crossref PubMed Scopus (144) Google Scholar) can be sulfated to alter biological activity. Growth factors and blood coagulation factors become fully activated after binding to glucosaminoglycan sulfates such as heparan sulfates (7Rosenberg R.D. Shworak N.W. Liu J. Schwartz J.J. Zhang L. J. Clin. Invest. 1997; 99: 2062-2070Crossref PubMed Scopus (254) Google Scholar, 8Nelson R.M. Venot A. Bevilacqua M.P. Linhardt R.J. Stamenkovic I. Annu. Rev. Cell Dev. Biol. 1995; 11: 601-631Crossref PubMed Scopus (101) Google Scholar). The sulfation/desulfation balance, thus, regulates cell growth and differentiation as well as neuronal and hormonal homeostasis. Sulfation generally detoxifies xenobiotics such as pharmaceutical drugs and synthetic and naturally occurring chemicals (9Falany C.N. FASEB J. 1997; 11: 206-216Crossref PubMed Scopus (517) Google Scholar). In some cases, however, xenobitic sulfation can be used for the in vivoactivation of prodrugs or can result in potentiating toxicity and carcinogenicity (10Meisheri K. Cipkus L.A. Taylor C.J. J. Pharmacol. Exp. Ther. 1987; 245: 751-760Google Scholar, 11Glatt H. FASEB J. 1997; 11: 314-321Crossref PubMed Scopus (184) Google Scholar). Sulfation of these diverse substrates is catalyzed by a group of enzymes referred to as sulfotransferases that use adenosine 3′-phosphate 5′-phosphosulfate (PAPS)1 as the ubiquitous sulfate donor. The crystal structure of the mouse estrogen sulfotransferase (EST) was the first three-dimensional structure to be solved for the sulfotransferase family (12Kakuta Y. Pedersen L.C. Chae K. Song W.-C. Leblanc D. London R. Cater C.W. Negishi M. Biochem. Pharmacol. 1998; 55: 313-317Crossref PubMed Scopus (27) Google Scholar, 13Kakuta Y. Pedersen L.G. Carter C.W. Negishi M. Pedersen L.C. Nat. Struct. Biol. 1997; 4: 904-908Crossref PubMed Scopus (231) Google Scholar). The structure revealed the catalytic core of the enzyme to be remarkably similar to those of nucleotide kinases, despite its little to no sequence homology to the kinases. The 5′-phosphate of the inactive cofactor adenosine 3′,5′-diphosphate (PAP) and the 3α-phenol group of estradiol (E2) in the EST·PAP·E2 structure superimpose well to the leaving β-phosphate group of ADP and the entering phosphate group of AMP in the UK-ADP-AMP structure, respectively (13Kakuta Y. Pedersen L.G. Carter C.W. Negishi M. Pedersen L.C. Nat. Struct. Biol. 1997; 4: 904-908Crossref PubMed Scopus (231) Google Scholar, 14Nuller-Dieckmann H. Schulz G.E.J. J. Mol. Biol. 1994; 236: 361-367Crossref PubMed Scopus (80) Google Scholar). These structural relationships, as well as an earlier study with phenol sulfotransferase (15Duffel M.W. Jakoby W.B. J. Biol. Chem. 1981; 256: 11123-11127Abstract Full Text PDF PubMed Google Scholar), suggest that the catalytic mechanism of sulfuryl and phosphoryl transfers may be similar. The lack of transition state information on the EST structure has made it difficult to visualize the sulfuryl transfer mechanism. The EST·PAP·E2 structure previously identified Lys48, Thr45, Thr51, The52, Lys106, His108, and Tyr240 as possible catalytic residues. We have performed site-directed mutagenesis studies on these residues in order to better understand the sulfuryl transfer mechanism of EST, and the roles of these residues in catalysis. Moreover, vanadate has been employed in crystallographic analysis in order to obtain a model of the transition state of EST. Based on these studies, we have identified residues involved in stabilizing the transition state that promotes the sulfuryl transfer reaction. We thank Rick Moore for excellent technical assistance. Our sincere appreciation is due to Drs. Lee Pedersen, William Beard, and Mike Duffel for helpful discussion.