Potent inhibition of human sulfotransferase 1A1 by 17α-ethinylestradiol: role of 3'-phosphoadenosine 5'-phosphosulfate binding and structural rearrangements in regulating inhibition and activity.

Sulfotransferase (SULT) 1A1 is the major drug/xenobiotic-conjugating SULT isoform in human liver because of its broad substrate reactivity and high expression level. SULT1A1 sulfates estrogens with low micromolar K(m) values consistent with its affinity for sulfation of many small phenolic compounds. Binding studies showed the unexpected ability of 17α-ethinylestradiol (EE2) to bind and inhibit SULT1A1 activity toward p-nitrophenol and ß-naphthol at low nanomolar concentrations, whereas EE2 was not sulfated until significantly higher concentrations were reached. EE2 had a K(i) of 10 nM for inhibiting p-nitrophenol and ß-naphthol sulfation and inhibited 17ß-estradiol (E2) sulfation in intact human MCF-7 breast cancer cells with a K(i) of 19 nM. In contrast, the K(m) for EE2 sulfation by SULT1A1 was 700 nM. The K(d) for EE2 binding of pure SULT1A1 was 0.5 ± 0.15 µM; however, the K(d) for EE2 binding to the SULT1A1-PAP complex was >100-fold lower (4.3 ± 1.7 nM). The K(d) for E2 binding to SULT1A1 changed from 2.3 ± 0.9 to 1.2 ± 0.56 µM in the presence of PAP. Docking studies with E2 indicate that E2 binds in a competent orientation in the resolved structure of SULT1A1 in the both presence and absence of 3'-phosphoadenosine 5'-phosphosulfate (PAPS). However, EE2 binds in a catalytically competent orientation in the absence of PAPS but in a noncompetent orientation via formation of a charge interaction with Tyr108 if PAPS is bound first. In conclusion, EE2 is a potent inhibitor, but not a substrate, of SULT1A1 at low nanomolar concentrations, indicating the possibility of drug-drug interactions during contraceptive therapy.

Sulfation of fulvestrant by human liver cytosols and recombinant SULT1A1 and SULT1E1.

Fulvestrant (Faslodex™) is a pure antiestrogen that is approved to treat hormone receptor-positive metastatic breast cancer in postmenopausal women. Previous studies have demonstrated that fulvestrant metabolism in humans involves cytochromes P450 and UDP-glucuronosyltransferases (UGTs). To date, fulvestrant sulfation has not been characterized. This study examined fulvestrant sulfation with nine recombinant sulfotransferases and found that only SULT1A1 and SULT1E1 displayed catalytic activity toward this substrate, with K(m) of 4.2 ± 0.99 and 0.2 ± 0.16 µM, respectively. In vitro assays of 104 human liver cytosols revealed marked individual variability that was highly correlated with ß-naphthol sulfation (SULT1A1 diagnostic substrate; r = 0.98, P < 0.0001), but not with 17ß-estradiol sulfation (SULT1E1 diagnostic substrate; r = 0.16, P = 0.10). Fulvestrant sulfation was correlated with both SULT1A1*1/2 genotype (P value = 0.023) and copy number (P < 0.0001). These studies suggest that factors influencing SULT1A1/1E1 tissue expression and/or enzymatic activity could influence the efficacy of fulvestrant therapy.

Lack of substrate inhibition in a monomeric form of human cytosolic SULT2A1.

Mammalian cytosolic sulfotransferases (SULTs) frequently show substrate inhibition during the sulfation of increasing concentrations of substrates. SULT2A1, a major human liver isoform responsible for the conjugation of hydroxysteroids, bile acids and aliphatic hydroxyl groups in drugs and xenobiotics, is a homodimer and displays substrate inhibition during the conjugation of dehydroepiandrosterone (DHEA). Maltose binding protein (MBP)-SULT2A1 fusion protein, produced as an intermediate step in the purification of the SULT2A1 homodimer, elutes during size exclusion chromatography as a monomer. The initial-rate parameters (Km and Vmax) of the monomer (MBP-SULT2A1) and native SULT2A1 dimer for DHEA sulfation are extremely similar; however, the monomer is not inhibited by DHEA. Intrinsic fluorescence studies show that two DHEA molecules bind each SULT2A1 subunit, one in the catalytic site and one in an apparent allosteric site. Lack of dimerization in the MBP-SULT2A1 fusion protein decreased the Kd for binding of DHEA at the allosteric site. These results suggest that formation of the homodimer is associated with structural re-arrangements leading to increased DHEA binding at an allo-steric site that is associated with substrate inhibition.

Structural rearrangement of SULT2A1: effects on dehydroepiandrosterone and raloxifene sulfation.

BACKGROUND: Human cytosoloic sulfotransferase (SULT) 2A1 is a major hepatic isoform and sulfates hydroxyl groups in structurally diverse sterols and xenobiotics. SULT2A1 crystal structures resolved in the presence and absence of 3',5'-diphosphoadenosine (PAP) or dehydropeiandrosterone (DHEA) suggest a significant rearrangement of the peptide that forms the surface of the active site in the presence of PAP. MATERIALS AND METHODS: Molecular modeling was used to examine the effects of the rearrangement in SULT2A1 associated with 3'-phosphoadenosine 5'-phosphosulfate (PAPS) binding on the binding of DHEA and raloxifene. The kinetics of DHEA and raloxifene sulfation was analyzed to investigate the effects of the rearrangement on SULT2A1 activity. RESULTS: Molecular models indicate that DHEA is able to bind to SULT2A1 in both conformations (open, without PAP; closed, with PAP) in a catalytic configuration, whereas raloxifene bound in a catalytic conformation only in the open structure. Raloxifene did not bind in the smaller, closed substrate binding pocket. Kinetic analysis of DHEA sulfation was consistent with a random Bi-Bi reaction mechanism, whereas raloxifene sulfation was more indicative of an ordered reaction mechanism with raloxifene binding first. Initial burst kinetics with DHEA yielded similar results after preincubation of SULT2A1 with DHEA or PAPS. Preincubation of SULT2A1 with raloxifene showed a burst of raloxifene sulfate formation with the addition of PAPS. In contrast, little raloxifene sulfate was formed if SULT2A1 was preincubated with PAPS and the reaction initiated with raloxifene. CONCLUSIONS: The structural rearrangements in SULT2A1 caused by PAPS binding can alter the sulfation mechanism and kinetics of different substrates.

Lack of substrate inhibition in a monomeric form of human cytosolic SULT2A1.

Mammalian cytosolic sulfotransferases (SULTs) frequently show substrate inhibition during the sulfation of increasing concentrations of substrates. SULT2A1, a major human liver isoform responsible for the conjugation of hydroxysteroids, bile acids and aliphatic hydroxyl groups in drugs and xenobiotics, is a homodimer and displays substrate inhibition during the conjugation of dehydroepiandrosterone (DHEA). Maltose binding protein (MBP)-SULT2A1 fusion protein, produced as an intermediate step in the purification of the SULT2A1 homodimer, elutes during size exclusion chromatography as a monomer. The initial-rate parameters (K(m) and V(max)) of the monomer (MBP-SULT2A1) and native SULT2A1 dimer for DHEA sulfation are extremely similar; however, the monomer is not inhibited by DHEA. Intrinsic fluorescence studies show that two DHEA molecules bind each SULT2A1 subunit, one in the catalytic site and one in an apparent allosteric site. Lack of dimerization in the MBP-SULT2A1 fusion protein decreased the K(d) for binding of DHEA at the allosteric site. These results suggest that formation of the homodimer is associated with structural rearrangements leading to increased DHEA binding at an allosteric site that is associated with substrate inhibition.

24-hydroxycholesterol sulfation by human cytosolic sulfotransferases: formation of monosulfates and disulfates, molecular modeling, sulfatase sensitivity, and inhibition of liver x receptor activation.

24-Hydroxycholesterol (24-OHChol) is a major cholesterol metabolite and the form in which cholesterol is secreted from the brain. 24-OHChol is transported by apolipoprotein E to the liver and converted into bile acids or excreted. In both brain and liver, 24-OHChol is a liver X receptor (LXR) agonist and has an important role in cholesterol homeostasis. 24-OHChol sulfation was examined to understand its role in 24-OHChol metabolism and its effect on LXR activation. 24-OHChol was conjugated by three isoforms of human cytosolic sulfotransferase (SULT). SULT2A1 and SULT1E1 sulfated both the 3- and 24-hydroxyls to form the 24-OHChol-3, 24-disulfate. SULT2B1b formed only 24-OHChol-3-sulfate. The 3-sulfate as a monosulfate or as the disulfate was hydrolyzed by human placental steroid sulfatase, whereas the 24-sulfate was resistant. At physiological 24-OHChol concentrations, SULT2A1 formed the 3-monosulfate and the 3, 24-disulfate as a result of a high affinity for sulfation of the 3-OH in 24-OHChol-24-sulfate. Molecular docking simulations indicate that 24-OHChol-24-sulfate binds in an active configuration in the SULT2A1 substrate binding site with high affinity only when the SULT2A1 homodimer structure was used. 24-OHChol is an LXR activator. In contrast, the 24-OHChol monosulfates were not LXR agonists in a fluorescence resonance energy transfer coactivator recruitment assay. However, both the 24-OHChol-3-sulfate and 24-sulfate were antagonists of LXR activation by N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trif-luoro-1-hydroxy-1-(trifluoromethyl)ethyl]phenyl]-benzenesulfonamide (T0901317) with an IC(50) of 0.15 and 0.31 muM, respectively. Inhibition of LXR activation by the 24-OHChol monosulfates at low nanomolar concentrations indicates that sulfation has a role in LXR regulation by oxysterols.