Abstract
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. (2008) Biochemistry 47, 11894-11899). On the basis of activation of SULT1A1 by para-nitrophenyl sulfate (pNPS), an ordered bypass mechanism has been proposed where pNPS sulfates PAP prior to its release from the E.PAP complex regenerating E.PAPS. Data are consistent with uncompetitive substrate inhibition by naphthol as a result of formation of the E.PAP.naphthol dead-end complex; formation of the complex is corroborated by naphthol/PAP double inhibition experiments. pNPS activation data demonstrate an apparent ping-pong behavior with pNPS adding to E.PAP, and competitive inhibition by naphthol consistent with formation of the E.PAP.naphthol complex. Exchange against forward reaction flux (PAPS plus naphthol) beginning with [35S]PAPS and generating [35S]naphthyl sulfate is also consistent with pNPS intercepting the E.PAP complex. Overall, data are consistent with the proposed ordered bypass mechanism.
Highlights
Containing xenobiotics [1,2,3,4,5,6,7,8]
On the basis of isotope exchange at equilibrium, we showed that the kinetic mechanism for human SULT1A1 is steady-state-ordered with phosphate 5Ј-phosphosulfate (PAPS) binding to the protein first, and PAP released last [13]
Data are indicative of uncompetitive substrate inhibition, consistent with the ordered kinetic mechanism proposed for SULT1A1 previously [13]
Summary
Containing xenobiotics [1,2,3,4,5,6,7,8]. 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. Human phenol sulfotransferase (SULT1A1) is one of the major detoxifying enzymes for phenolic xenobiotics; it catalyzes the sulfation of endogenous hydroxyl biosignaling molecules. It has very broad substrate specificity and high activity toward most phenolic compounds. A ternary complex formed between substrate and the enzyme1⁄7PAP complex is the most likely possibility in an ordered mechanism, but binding to free enzyme is possible [12, 16]. Binding of two substrate molecules to the active site has been proposed [4, 14]. An additional EAP dead-end complex is allowed but not shown
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