CATALYTIC MECHANISM OF HUMAN SULFOTRANSFERASE 1A1

Abstract

Cytosolic sulfotransferases (SULTs) catalyzed sulfation regulates hormones and detoxifies 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). Human SULT1A1 catalyzes the sulfation of xenobiotic phenols and has very broad substrate specificity. Based on our recent data, we propose an ordered sequential Bi Bi mechanism for SULT-catalyzed reactions with either binary complex (E:S) formation or ternary complex (E:PAP:S) formation leading to substrate inhibition. Based on the activation of this sulfation by a second product, p-nitrophenylsulfate (pNPS), we proposed a bypass ordered mechanism for human SULT1A1 catalyzed simple phenol sulfation. The complete rate equations were derived using the Indge and Childs method. Data fitting results suggest that substrate inhibition of human SULT1A1 occurs via the formation of an E:PAP:S but not E:S complex. A characteristic set of straight lines in the double-reciprocal plot of 1/V vs. 1/[PAPS] at low substrate concentrations points to an ordered Bi Bi mechanism with the PAPS binding first. Lack of inhibition by PAPS supports this conclusion. Furthermore, uncompetitive inhibition by 2-naphthol at high substrate concentrations supports the ordered Bi Bi mechanism, and is in accordance with the rate equation derived for E:PAP:S formation. At saturating levels of PAPS and varying 2-naphthol and pNPS conditions, observed kinetics for the bypass reaction agrees well with our proposed bypass ordered mechanism. Competitive inhibition by 2-naphthol at high substrate concentrations is in agreement with the derived bypass rate equation.