Isotope Exchange at Equilibrium Indicates a Steady State Ordered Kinetic Mechanism for Human Sulfotransferase

Abstract

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

para-Nitrophenyl Sulfate Activation of Human Sulfotransferase 1A1 Is Consistent with Intercepting the E·PAP Complex and Reformation of E·PAPS

“…The slope is a linear function of 1/naphthol, whereas the intercept replot exhibits substrate inhibition as an increase in the intercept as 1/naphthol approaches zero. Data are indicative of uncompetitive substrate inhibition, consistent with the ordered kinetic mechanism proposed for SULT1A1 previously (13). A fit of the data to Equation 1 gives the kinetic parameters in Table 1.…”

“…Kinetic Mechanism of SULT1A1-Previous studies, using isotope exchange at equilibrium, suggested the kinetic mechanism of human SULT1A1 is a steady state ordered with PAPS binding to the enzyme first (13). Uncompetitive substrate inhibition by naphthol is consistent with a bi-bi ordered sequential mechanism (34,35), with naphthol binding to the E⅐PAP product complex, slowing the release of PAP.…”

“…The K i for substrate inhibition by naphthol is an order of magnitude higher than its K m . Double Inhibition by PAP and Naphthol-Previous data suggested an ordered mechanism for SULT1A1 with PAPS binding to the enzyme first followed by substrate (13). In this mechanism, uncompetitive substrate inhibition by naphthol is common and suggests slow release of last product (PAP), but binding to central complexes is also possible (32).…”

“…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).…”

“…Double Inhibition Experiments-The initial concentration of PAPS was 5 M (ϳ5 times K PAPS ) (13). The initial concentrations of PAP were 0, 5, 10, 15, and 20 M. A stock 1-naphthol solution was made in 100% ethanol and doped with 14 C-labeled 1-naphthol.…”