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

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 center dot PAP complex regenerating E center dot PAPS. Data are consistent with uncompetitive substrate inhibition by naphthol as a result of formation of the E center dot PAP center dot 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 center dot PAP, and competitive inhibition by naphthol consistent with formation of the E center dot PAP center dot naphthol complex. Exchange against forward reaction flux (PAPS plus naphthol) beginning with [S-35] PAPS and generating [S-35] naphthyl sulfate is also consistent with pNPS intercepting the E center dot PAP complex. Overall, data are consistent with the proposed ordered bypass mechanism.