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

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. 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...

Design and Interpretation of Human Sulfotransferase A1 Assays

“…PAPS and raloxifene parameter files were generated using PQS (Parallel Quantum Solutions, Fayetteville, AR). (Duffel and Jakoby, 1981;Tyapochkin et al, 2009;Cook et al, 2015a) and may explain the apparent discrepancy in catalytic efficiency of fulvestrant toward SULT1A1 (Edavana et al, 2011;Cook et al, 2013a). The PAPS concentrations in human tissues are such that the specificity of SULT1A1 is expected to be highly tissue dependent.…”

“…The SULTs typically exhibit partial substrate inhibition, a type of inhibition in which turnover decreases to a nonzero value at saturating substrate. Several mechanisms have been proposed to explain this inhibition in SULTs, including an allostericbinding pocket (Zhang et al, 1998), gating (Lu et al, 2008;Cook et al, 2010), the binding of multiple acceptors in the active site (Gamage et al, 2003), and the formation of a dead-end complex (Gamage et al, 2005;Tyapochkin et al, 2009;Gulcan and Duffel, 2011;Wang et al, 2014a). In a recent study of SULT2A1, each of the 22 microscopic rate constants associated with interconversion of the 11 complexes in the mechanism were determined (Wang et al, 2014a).…”

Mechanism of sulfotransferase pharmacogenetics in altered xenobiotic metabolism

“…This is because when the alternate activated sulfate source (such as pNPS) is used, the release of PAP, which is the rate-limiting step, is not required to continue the next round of sulfation reaction. However, substrate inhibition continues to exist [80], and competitive inhibition with respect to sulfated substrate can be observed. In such sulfation reactions, PAP serves as a cofactor of SULT.…”

“…As shown in Figure 5, under a high substrate concentration, the substrate S may combine with [E:PAP] and form the dead-end complex [E:PAP:S] [78] to exhibit substrate inhibition. The [E:PAP:S] complex (SULT1A1/PAP/E2, PDB code: 2D06; SULT2B1b/PAP/DHEA, PDB code: 1Q22) [56,79] structural analysis and kinetic studies have suggested that substrate inhibition most likely causes the ternary complex formation [78,80,81]. The product or the sulfated-substrate (sS) with the [E:PAPS] binary complex can form a dead-end ternary complex [73].…”

Structure, dynamics and selectivity in the sulfotransferase family

“…Despite its apparent simplicity, the mechanistic underpinnings of this curve-shape have been debated for more than three decades (Duffel & Jakoby, 1981). Explanations include subunit interactions (Petrotchenko et al, 2001), non-productive binding, allosteric binding (Lu et al, 2008), multiple active-site substrates (Gamage et al, 2003) and dead-end complex formation (Gulcan & Duffel, 2011; Sun & Leyh, 2010; Tyapochkin et al, 2009). Here we consider for the first time, that gating of the cap is fundamental to substrate inhibition and highlight recent findings that support this concept.…”