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It is known that in humans taking soy food, the phytoestrogens, daidzein (DZ) and genistein (GS), exist as sulfates and glucuronides in the plasma and are excreted as conjugates in urine. To investigate which human sulfotransferase (SULT) isoforms participate in the sulfation of these phytoestrogens, the four major cytosolic SULTs, SULT1A1, SULT1A3, SULT1E1, and SULT2A1, occurring in the human liver were bacterially expressed as His-tagged proteins and chromatographically purified to homogeneity in the presence of Tween 20 and glycerol as highly efficient agents for stabilizing the recombinant enzymes. All the SULTs showed sulfating activity toward both DZ and GS. However, k(cat)/K(m) values observed indicated that these phytoestrogens were sulfated predominantly by SULT1A1 and SULT1E1 with K(m) values of 0.3 and 0.7 microM for GS and 1.9 and 3.4 microM for DZ, respectively. DZ and GS strongly inhibited the sulfation of the endogenous substrate, beta-estradiol, by SULT1E1 in a non-competitive manner with K(i) values of 14 and 7 microM, respectively, suggesting that these phytoestrogens might affect tissue levels of beta-estradiol in the human. The phenolic endocrine-disrupting chemicals, bisphenol A (BPA), 4-n-nonylphenol (NP), and 4-t-octylphenol (t-OP), were used as substrates to investigate the possible participation of human SULTs in their metabolism for excretion. High k(cat)/K(m) values were observed for the sulfation of BPA by SULT1A1, NP by SULT1A1 and SULT1E1, and t-OP by SULT1E1 and SULT2A1.

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Regioselective sulfation of the phytoestrogens daidzein (DZ, 7,4'-dihydroxyisoflavone) and genistein (GS, 5,7,4'-trihydroxyisoflavone) was investigated using human liver cytosol and purified recombinant human sulfotransferase (SULT) isoforms, SULT1A1, SULT1A3, SULT2A1, and SULT1E1. 7-Position-preferential sulfation of DZ and GS was observed in human hepatic cytosols from 3 male and 3 female subjects. Average ratios for 7- to 4'-sulfate formation were 4.5:1 from DZ and 8.4:1 from GS in these human liver cytosols. Apparent K(m) values for the 7- and 4'-sulfation of DZ and GS by these cytosols were similar and in a range from 0.46 to 0.66 microM. All recombinant human SULTs had activity for 7- and 4'-sulfation of these phytoestrogens except for 7-sulfating activity of SULT1A3. SULT1A1 and SULT1E1 exhibited much higher catalytic efficiency, k(cat)/K(m), for 7- and 4'-sulfation of these substrates than did the other two, SULT1A3 and SULT2A1. SULT1A1 showed K(m) values of 0.47 and 0.52 microM for the mono-sulfation of DZ and GS, respectively, which were very similar to those of human cytosol. The observed k(cat)/K(m) indicated that SULT1A1 catalyzed 7-sulfation of DZ and GS at rates 4.4- and 8.8-fold higher, respectively, than such 4'-sulfation. However, with SULT1E1, catalytic efficiency was very similar for the sulfation of both positions. These data strongly suggest that SULT1A1 plays a major role in monosulfation of the phytoestrogens and determines the regioselectivity of sulfation in human hepatic cytosol. A kinetic study for 7,4'-disulfate formation of DZ and GS from their 7- and 4'-monosulfates indicated that SULT1E1 most efficiently catalyzed both reactions among human SULTs.