The Allosteric Binding Sites of Sulfotransferase 1A1

Abstract

Human sulfotransferases (SULTs) comprise a small, 13-member enzyme family that regulates the activities of thousands of compoundsendogenous metabolites, drugs, and other xenobiotics. SULTs transfer the sulfuryl-moiety (-SO 3 ) from a nucleotide donor, PAPS (39-phosphoadenosine 59-phosphosulfate), to the hydroxyls and primary amines of acceptors. SULT1A1, a progenitor of the family, has evolved to sulfonate compounds that are remarkably structurally diverse. SULT1A1, which is found in many tissues, is the predominant SULT in liver, where it is a major component of phase II metabolism. Early work demonstrated that catechins and nonsteroidal antiinflammatory drugs inhibit SULT1A1 and suggested that the inhibition was not competitive versus substrates. Here, the mechanism of inhibition of a single, high affinity representative from each class [epigallocatechin gallate (EGCG) and mefenamic acid] is determined using initial-rate and equilibrium-binding studies. The findings reveal that the inhibitors bind at sites separate from those of substrates, and at saturation turnover of the enzyme is reduced to a nonzero value. Further, the EGCG inhibition patterns suggest a molecular explanation for its isozyme specificity. Remarkably, the inhibitors bind at sites that are separate from one another, and binding at one site does not affect affinity at the other. For the first time, it is clear that SULT1A1 is allosterically regulated, and that it contains at least two, functionally distinct allosteric sites, each of which responds to a different class of compounds.

The structure of the catechin-binding site of human sulfotransferase 1A1

“…3B), which is consistent with the fact that the affinities of acceptors small enough to pass through the pore are not affected by its presence (22). The cap closes 18-fold more "tightly" when EGCG is bound (11,41); indeed, the enhanced trapping of nucleotide that results from this tighter binding appears to be the mechanistic basis of EGCG inhibition (41). Here, we see the structural basis for this inhibition-the rings of EGCG are entwined with those of the lip and cap (Fig.…”

“…Initial-rate and epigallocatechin gallate (EGCG)-inhibition parameters for the labeled constructs were determined using a previously described PNP assay (11). Briefly, reactions are initiated by addition of 0.50 mM PAPS (17 × K m ) to a solution containing 0.10 μM enzyme, 10 μM PNP (∼16 × K m ), and 50 mM KPO 4 (pH 7.5; 25 ± 2°C).…”

“…Early screening studies intimated that SULTs might be subject to small-molecule allosteric control (9,10), and recent work confirms this (11). SULT1A1, the most abundant isoform in adult human liver, harbors at least two physically separate and noninteracting allosteric-binding sites, each of which binds a different complex class of molecules-the catechins (abundant flavonoids in tea and cacao) (9) and nonsteroidal antiinflammatory drugs (NSAIDs) (10).…”

“…EGCG is, however, unique in that it exhibits particularly high affinity (K i ∼30 nM) for a single isoform, SULT1A1 (9,11). To date, there are no published structures of SULT-bound allosteres.…”

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.…”