Conjugation of many xenobiotics, drugs, and endogenous compounds with a sulfonate moiety is an important reaction in their biotransformation. Sulfation of these compounds generally results in a decrease in biological activity and an increase in their urinary excretion. However, in certain instances, sulfation results in bioactivation to reactive electrophilic or therapeutically active forms. At least four cytosolic sulfotransferases (STs) have been identified and characterized from human tissues. These enzymes are two forms of phenol ST (PST), the phenol-sulfating and the monoamine-sulfating forms of PST (P-PST and M-PST, respectively), an estrogen sulfotransferase (EST), and a hydroxysteroid ST, dehydroepiandrosterone ST (DHEA-ST). Although four cytosolic STs have been well characterized in human tissues, evidence is accumulating for the presence of allelic forms or additional distinct forms of the STs in human tissues. The STs possess distinct but overlapping substrate specificities, and all of the STs are capable of conjugating both xenobiotic and endogenous compounds. The individual forms of ST may display distinct patterns of tissue specific expression and different mechanisms of regulation. Although the role of sulfation in drug metabolism is well recognized, an increased understanding of the biochemistry and molecular biology of the STs should also provide additional information as to their functions in many normal physiologic processes.

Estrogen sulfotransferase (EST) catalyzes the transfer of the sulfuryl group from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to 17beta-estradiol (E2). The sulfation of E2 prevents it from binding to, and thereby activating, the estrogen receptor. The regulation of EST appears to be causally linked to tumorigenesis in the breast and endometrium. In this study, recombinant human EST is characterized, and the catalytic mechanism of the transfer reaction is investigated in ligand binding and initial rate experiments. The native enzyme is a dimer of 35-kDa subunits. The apparent equilibrium constant for transfer to E2 is (4.5 +/- 0.2) x 10(3) at pH 6.3 and T = 25 +/- 2 degrees C. Initial rate studies provide the kinetic constants for the reaction and suggest a sequential mechanism. E2 is a partial substrate inhibitor (Ki = 80 +/- 5 nM). The binding of two E2 per EST subunit suggests that the partial inhibition occurs through binding at an allosteric site. In addition to providing the dissociation constants for the ligand-enzyme complexes, binding studies demonstrate that each substrate binds independently to the enzyme and that both the E.PAP.E2S and E.PAP.E2 dead-end complexes form. These results strongly suggest a Random Bi Bi mechanism with two dead-end complexes.

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Human cytosolic sulfotransferases (SULTs) transfer the sulfuryl-moiety (-SO3) from activated sulfate (3′-phosphoadenosine 5′-phosphosulfate, PAPS) to the hydroxyls and primary amines of numerous metabolites, drugs and xenobiotics. Receipt of the sulfuryl-group often radically alters acceptor-target interactions. How these enzymes select particular substrates from the hundreds of candidates in a complex cytosol remains an important question. Recent work reveals PAPS binding causes SULT2A1to undergo an isomerization that controls selectivity by constricting the opening through which acceptors must pass to enter the active site. The enzyme maintains an affinity for large substrates by isomerizing between the open and closed states with nucleotide bound. Here, the molecular basis of the nucleotide-induced closure is explored in equilibrium and non-equilibrium molecular dynamics simulations. The simulations predict that the active-site “cap,” which covers both the nucleotide and acceptor binding sites, opens and closes in response to nucleotide. The cap subdivides into nucleotide and acceptor halves whose motions, while coupled, exhibit an independence that can explain the isomerization. In-silico weakening of electrostatic interactions between the cap and base of the active site causes the acceptor-half of the cap to open and close while the nucleotide lid remains shut. Simulations predict that SULT1A1, the most abundant SULT in human liver, will utilize a similar selection mechanism. This prediction is tested using fulvestrant, an antiestrogen too large to pass through the closed pore, and estradiol, which is not restricted by closure. Equilibrium and presteady state binding studies confirm that SULT1A1 undergoes a nucleotide induced isomerzation that controls substrate selection.

A form of sulphotransferase capable of sulphating dehydroepiandrosterone and other steroids was purified from cytosol prepared from human liver. Dehydroepiandrosterone sulphotransferase was purified 621-fold when compared with the activity in cytosol using DEAE-Sepharose CL-6B and adenosine 3',5'-bisphosphate-agarose affinity chromatography. During affinity chromatography, dehydroepiandrosterone sulphation activity could be resolved from p-nitrophenol sulphation activity catalysed by phenol sulphotransferase by using a gradient of adenosine 3'-phosphate 5'-phosphosulphate. The purified enzyme was most active towards dehydroepiandrosterone but was capable of conjugating a number of other steroids, including pregnenolone, androsterone and beta-oestradiol. No activity towards p-nitrophenol or dopamine, substrates for the phenol sulphotransferase, was observed with the pure enzyme. A single band with a subunit molecular mass of 35 kDa was observed by Coomassie Blue staining following SDS/polyacrylamide-gel electrophoresis of the purified enzyme. A molecular mass of 68-70 kDa was calculated for the active form of the enzyme by chromatography on Sephacryl S-200, suggesting that the active form of the enzyme is a dimer.

ABSTRACT:Triclosan is a broad spectrum antibacterial agent used in many household products. Due to its structural similarity to polychlorobiphenylols, which are potent inhibitors of the sulfonation and glucuronidation of 3-hydroxy-benzo[a]pyrene, it was hypothesized that triclosan would inhibit these phase II enzymes. This study was designed to assess the interactions of triclosan as a substrate and inhibitor of 3-phosphoadenosine 5-phosphosulfate-sulfotransferases and UDP-glucuronosyltransferases in human liver cytosol and microsomes.

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Human SULT2A1 is one of two predominant sulfotransferases in liver and catalyzes transfer of the sulfuryl moiety (−SO 3 ) from activated sulfate (PAPS, 3′-phosphoadenosine 5-phosphosulfate) to hundreds of acceptors (metabolites and xenobiotics). Sulfation recodes the biologic activity of acceptors by altering their receptor interactions. The molecular basis on which these enzymes select and sulfonate specific acceptors from complex mixtures of competitors in vivo is a long-standing issue in the SULT field. Raloxifene, a synthetic steroid used in the prevention of osteoporosis, and dehydroepiandrosterone (DHEA), a ubiquitous steroid precusor, are reported to be sulfated efficiently by SULT2A1 in vitro, yet unlike DHEA, raloxifene is not sulfated in vivo. This selectivity was explored in initial rate and equilibrium binding studies that demonstrate pronounced binding antisynergy (21-fold) between PAPS and raloxifene, but not DHEA. Analysis of crystal structures suggests that PAP binding restricts access to the acceptor-binding pocket by restructuring a nine-residue segment of the pocket edge that constricts the active site opening, or "pore", that sieves substrates on the basis of their geometries. In silico docking predicts that raloxifene, which is considerably larger than DHEA, can bind only to the unliganded (open) enzyme, whereas DHEA binds both the open and closed forms. The predictions of these structures with regard to substrate binding are tested using equilibrium and pre-steady-state ligand binding studies, and the results confirm that a nucleotide-driven isomerization controls access to the acceptor-binding pocket and plays an important role in substrate selection by SULT2A1 and possibly other sulfotransferases.