Cytosolic sulfotransferases (SULTs)-catalyzed sulfation regulates bio-signaling molecule biological activities and detoxifies hydroxyl-containing xenobiotics. The universal sulfuryl group donor for SULT-catalyzed sulfation is adenosine 3′-phosphate 5′-phosphosulfate (PAPS). The reaction products are a sulfated product and adenosine 3′, 5′-diphosphate (PAP). Although the kinetics has been reported since the 1980's, SULTs-catalyzed reaction mechanisms remain unclear. Human SULT1A1 catalyzes the sulfation of xenobiotic phenols and has very broad substrate specificity. It has been recognized as one of the most important phase II drug metabolizing enzymes. Understanding of the kinetic mechanism of this isoform is important for the understanding of drug metabolism and xenobiotic detoxification. In this report, we investigated the SULT1A1-catalyzed phenol sulfation mechanism. The SULT1A1-catalyzed reaction was brought to equilibrium varying substrate (1-naphthol) and PAPS initial concentrations. Equilibrium constants were calculated. In addition, substrate/product equilibrium ratio (S e /P e ) was determined by perturbation of equilibrium, varying S/P ratios and measuring the shift in product amounts. Two isotopic exchanges at equilibrium: [ 14 C]1-naphthol ⇋ [ 14 C]1-naphthyl sulfate, and [ 35 S]PAPS ⇋ [ 35 S]1-naphthyl sulfate were measured. First order kinetics, observed for all the isotopic exchange reactions studied over the entire time scale monitored, indicates that the system was truly at equilibrium prior to adding isotopic pulse. Complete suppression of 35 S isotopic exchange rate upon increasing 1-naphthol and 1-naphthyl sulfate in constant ratio, while no suppression of 14 C exchange rate was observed upon increasing PAPS and PAP in constant ratio. Data are consistent with a steady-state ordered kinetic mechanism with PAPS and PAP binding to free enzyme. Sulfotransferases (SULTs) catalyze the sulfation of hydroxyl-containing compounds as shown in Scheme 1 (1-10). The universal sulfuryl group donor for SULT-catalyzed sulfation is adenosine 3′-phosphate 5′-phosphosulfate (PAPS). The reaction products are a sulfated product and adenosine 3′, 5′-diphosphate (PAP).Studies of SULT catalytic mechanisms began in the early 1980's. However, the kinetic mechanism of the sulfotransferases has not yet been clearly defined. Initial velocity studies of the rat aryl SULT (AST-IV), were consistent with either a rapid equilibrium random Bi Bi mechanism with two dead-end product complexes or an ordered Theorell-Chance mechanism (11). On the other hand, similar studies of the kinetic mechanism of the human SULT1E1 were

Cytosolic sulfotransferase (SULT)-catalyzed sulfation regulates biological activities of various biosignaling molecules and metabolizes hydroxyl-containing drugs and xenobiotics. The universal sulfuryl group donor for SULT-catalyzed sulfation is adenosine 3-phosphate 5-phosphosulfate (PAPS), whereas the reaction products are a sulfated product and adenosine 3,5-diphosphate (PAP). Although SULT-catalyzed kinetic mechanisms have been studied since the 1980s, they remain unclear. Human SULT1A1 is an important phase II drug-metabolizing enzyme. Previously, isotope exchange at equilibrium indicated steady-state ordered mechanism with PAPS and PAP binding to the free SULT1A1 (Tyapochkin, E., Cook, P. F., and Chen, G. Sulfotransferases (SULTs)3 are phase II drug-metabolizing enzymes that catalyze the sulfation (sulfonation) of various hydroxyl-containing compounds: biosignaling molecules such as hydroxysteroid hormones, thyroid hormones, glucocorticoid hormones, bile acids, neurotransmitters, and hydroxylcontaining xenobiotics (1-8). The sulfation proceeds as shown in reaction 1, where the sulfuryl group donor is adenosine 3Ј-phosphate 5Ј-phosphosulfate (PAPS), and the reaction products are adenosine 3Ј,5Ј-diphosphate (PAP) and a sulfated product. R-OH ϩ PAPS L | ;One of the main biological functions of SULTs is the regulation of various hormones (9). Sulfation of xenobiotics is mainly associated with detoxification, biotransformation of a relatively hydrophobic xenobiotic into a more water-soluble sulfuric ester that is readily excreted. However, in some cases sulfation can also cause bioactivation of procarcinogens and promutagens, leading to possible toxic effects (10, 11).Studies of the SULTs kinetic mechanisms began to appear in the early 1980s (12). Although many SULT isoforms have been isolated and characterized, their biological functions and catalytic mechanisms are still not well understood. Human phenol sulfotransferase (SULT1A1) is one of the major detoxifying enzymes for phenolic xenobiotics; it also catalyzes the sulfation of endogenous hydroxyl biosignaling molecules. It has very broad substrate specificity and high activity toward most phenolic compounds. SULT1A1 is also widely distributed in the human body. On the basis of isotope exchange at equilibrium, we showed that the kinetic mechanism for human SULT1A1 is steady-state-ordered with PAPS binding to the protein first, and PAP released last (13).Substrate inhibition by the hydroxyl substrate (sulfate acceptor) is a common feature of most cytosolic SULTs (14,15). Inhibition of SULT1A1 has been observed by the substrate, naphthol. There are a number of different mechanisms that have been proposed for substrate inhibition, but the mechanism remains unclear. A ternary complex formed between substrate and the enzyme⅐PAP complex is the most likely possibility in an ordered mechanism, but binding to free enzyme is also possible (12,16). It is also possible, but unlikely, that substrate could bind to central complexes. In addition, binding of two substrate...

Cytosolic sulfotransferase (SULT)-catalyzed sulfation regulates the activity of bio-signaling molecules and aids in metabolizing hydroxyl-containing xenobiotics. The sulfuryl donor for the SULT reaction is adenosine 3′-phosphate 5′-phosphosulfate (PAPS), while products are adenosine 3′,5′-diphosphate (PAP) and a sulfated alcohol. Human phenol sulfotransferase (SULT1A1) is one of the major detoxifying enzymes for phenolic xenobiotics. The mechanism of SULT1A1-catalyzed sulfation of PAP by pNPS was investigated. PAP was sulfated by para-nitrophenyl sulfate (pNPS) in a concentration-dependent manner. 2-Naphthol inhibited sulfation of PAP, competing with pNPS, while phenol activated the sulfation reaction. At saturating PAP, a ping pong kinetic mechanism is observed with pNPS and phenol as substrates, consistent with phenol intercepting the E–PAPS complex prior to dissociation of PAPS. At high concentrations, phenol competes with pNPS, consistent with formation of the E–PAP–phenol dead-end complex. Data are consistent with the previously reported mechanism for sulfation of 2-naphthol by PAPS, and its activation by pNPS [14]. Overall, data are consistent with release of PAP from E–PAP and PAPS from E–PAPS contributing to rate-limitation in both reaction directions.

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