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 compounds-endogenous metabolites, drugs, and other xenobiotics. SULTs transfer the sulfuryl-moiety (-SO3) from a nucleotide donor, PAPS (3'-phosphoadenosine 5'-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 anti-inflammatory 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.

Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Figures

Fig. 1.

The inhibition of SULT1A1 by EGCG. (A) EGCG versus PAPS. PAPS concentration was varied from 0.2 to 5 × Km, and EGCG concentrations are given in the figure. Reactions were initiated by addition of pNP at saturation (30 μM, 20 × Km), and reaction progress was monitored by following formation of 35S-pNPS. Velocities were determined by least-squares fitting of four-point progress curves. Less than 5% of the concentration-limiting substrate consumed at the reaction endpoint was converted during the measurement. Velocities were determined in duplicate, averaged, and the data were fit globally using an uncompetitive model. The results of the fit are given by the solid lines passing through the data. (B) EGCG versus pNP. Reactions were initiated by addition of PAPS at saturation (10 μM, 625 × Km), the pNP concentration varied from 0.2 to 5 × Km, and the EGCG concentration is given in the figure. Reaction progress was monitored at 405 nm. Less than 5% of the concentration-limiting substrate consumed at the endpoint of the reaction was converted during the measurement. Each point represents the average of three independent determinations. The lines through the points represent the behavior predicted by a global fit using a noncompetitive inhibition model. Reaction conditions for both panels were as follows: SULT1A1 (10 nM, dimer), MgCl2 (5.0 mM), and NaPO4 (50 mM), pH = 7.2, and T = 25 ± °C.

Fig. 2.

The binding of EGCG to SULT1A1. Binding was monitored via ligand-induced changes in the intrinsic fluorescence of SULT1A1 (λex = 295 nm, λem = 345 nm). Conditions were as follows: SULT1A1 (10 nM, dimer), PAP (0 or 10 μM, 33 × Kd), pNP (0 or 45 μM, 30 × Kd), MgCl2 (5.0 mM), NaPO4 (50 mM), pH 7.2, T = 25 ± 2°C. Each point is the average of two independent determinations. The line through the data represents a least-squares fit using a model that assumes a single binding site per subunit.

Fig. 3.

The mechanism of SULT1A1 inhibition. Inhibitor (EGCG or MEF) binds to each of the enzyme forms in the substrate portion of the catalytic cycle. Turnover (kcat) for the inhibited and noninhibited species are related by α.

Fig. 4.

The inhibition of SULT1A1 by MEF. (A) MEF versus PAPS. Protocols were nearly identical to those associated with Fig 1, A and B. PAPS concentration was varied from 0.2 to 5 × Km, and MEF concentrations are listed in the figure. Reactions were initiated by addition of pNP at saturation (30 μM, 20 × Km), and reaction progress was monitored by following formation of 35S-pNPS. Velocities, obtained by least-squares fitting of four-point progress curves, were determined in duplicate, averaged, and the data were fit globally using a noncompetitive model. The fitting results are given by lines passing through the data. (B) MEF versus pNP. Reactions were initiated by addition of PAPS at saturation (10 μM, 625 × Km). The pNP concentration varied from 0.2 to 5 × Km, and MEF concentrations are given in the figure. Reaction progress was monitored at 405 nm. Less than 5% of the concentration-limiting substrate consumed at the endpoint of the reaction was converted during the measurement. Each point represents the average of three independent determinations. The lines through the points represent the behavior predicted by a global fit using a noncompetitive inhibition model. Reaction conditions for both panels are as follows: SULT1A1 (5.0 nM, dimer), MgCl2 (5.0 mM), and NaPO4 (50 mM), pH = 7.2, and T = 25 ± °C.

Fig. 5.

The binding of MEF to SULT1A1. The protocol was virtually identical to that described in Fig 2. Binding was monitored via ligand-induced changes in the intrinsic fluorescence of SULT1A1 (λex = 295 nm, λem = 345 nm). Each point is the average of two independent determinations. The line through the data represents a least-squares fit using a model that assumes a single binding site per subunit. Conditions were as follows: SULT1A1 (10 nM, dimer), PAP (0 or 10 μM, 33 × Kd), pNP (0 or 45 μM, 30 × Kd), MgCl2 (5.0 mM), NaPO4 (50 mM), pH 7.2, T = 25 ± 2°C.

Fig. 6.

EGCG and MEF are partial inhibitors. Reaction progress was monitored at 405 nm. The conditions were as follows: SULT1A1 (1.0 nM, dimer), PAPS (10 μM, 625 × Km), PnP (30 μM, 22 × Km), MgCl2 (5.0 mM), NaPO4 (50 mM), pH 7.2, T = 25 ± 2°C. Less than 5% of the substrate converted at the endpoint of the reaction was consumed during the rate measurements. Each point represents the average of three independent determinations. The lines through the points indicate the behavior predicted by a least-squares fit using a model that assumes a single binding site per subunit. Ki values in the model were fixed using constants in Table 1, and data were fit only for the maximum inhibition value. The best-fit, maximum inhibition values for EGCG and MEF were 88 ± 2 and 94 ± 1%, respectively.

Fig. 7.

EGCG and MEF bind at separate and noninteracting sites. The pattern of SULT1A1 inhibition by EGCG and MEF in combination was used to assess their binding independence. EGCG and MEF were added simultaneously in equal Ki-equivalents over a concentration range that (as suggested by single-inhibitor studies) would cause the enzyme to transition from singly- to doubly-inhibitor bound. The curving solid lines are the predictions of a same-site (dashed line) and independent-site (solid line) binding models that were parameterized using the constants in Table 1. The experimental data (black dots) is in strong agreement with the additive model. Reaction conditions: SULT1A1 (20 nM), PAPS (10 μM, 625 × Km), pNP (36 μM, 22 × Km), MgCl2 (5.0 mM), and NaPO4 (50 mM), pH 7.2, T = 25 ± 2°C. Reactions were monitored at 405 nm. Each point is the average of three independent trials.