Paradigms of sulfotransferase catalysis: the mechanism of SULT2A1

Abstract

Human cytosolic sulfotransferases (SULTs) regulate the activities of thousands of signaling small molecules via transfer of the sulfuryl moiety (-SO3) from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to the hydroxyls and primary amines of acceptors. Sulfonation controls the affinities of ligands for their targets, and thereby regulates numerous receptors, which, in turn, regulate complex cellular responses. Despite their biological and medical relevance, basic SULT mechanism issues remain unresolved. To settle these issues, and to create an in-depth model of SULT catalysis, the complete kinetic mechanism of a representative member of the human SULT family, SULT2A1, was determined. The mechanism is composed of eight enzyme forms that interconvert via 22 rate constants, each of which was determined independently. The result is a complete quantitative description of the mechanism that accurately predicts complex enzymatic behavior. This is the first description of a SULT mechanism at this resolution, and it reveals numerous principles of SULT catalysis and resolves previously ambiguous issues. The structures and catalytic behaviors SULTs are highly conserved; hence, the mechanism presented here should prove paradigmatic for the family.

Keywords: DHEA; Enzyme Mechanism; Ligand-binding protein; Metabolic Regulation; Metabolism; Pre-steady State; Rate Constant; SULT2A1; Substrate Inhibition; Sulfotransferase.

FIGURE 1.

Pre-steady state binding of PAP to the SULT2A1·DHEA complex. A, the binding of PAP to SULT2A1·DHEA. Reactions were initiated by rapidly mixing (1:1 v/v) a solution containing PAP (0.25 μm, 1.0 × K_d [E·DHEA]) and DHEA (25 μm, 23 × K_d [E]) with a solution containing SULT2A1 (0.050 μm) and DHEA (25 μm). Binding was monitored by following changes in SULT2A fluorescence (λ_ex = 290 nm, and λ_em ≥ 330 nm, where ex represents excitation and em represents emission). Fluorescence intensity is plotted relative to the intensity at time 0 (I/I_o). Solutions contained MgCl₂ (5.0 mm), NaPO₄ (25 mm), pH 7.4, and were equilibrated at 25 ± 2 °C prior to mixing. The solid curve represents the best-fit behavior predicted by a single-exponential model. B, k_obs versus [PAP]. Reactions were pseudo-first order in PAP in all cases. Each point represents the average of three independent determinations. The solid line through the points represents a linear least square fit of the data. The slope and intercept of the line provide k_on and k_off, respectively.

FIGURE 2.

A burst of product. Reactions were initiated by rapidly mixing (1:1 v/v) a solution containing SULT2A1 (24 μm, dimer) with [³⁵S]PAPS (40 μm, 4.1 Ci/μmol, 133 × K_d [E]). The solution was then mixed with an equal volume of DHEA (50 μm, 46 × K_d [E·PAPS]); all solutions contained MgCl₂ (5.0 mm), NaPO₄ (50 mm), pH 7.2, and were equilibrated at 25 ± 2 °C prior to mixing. Reactions were quenched with NaOH (0.20 m, final), neutralized, boiled, and centrifuged to remove the protein. [³⁵S]DHEAS was separated from [³⁵S]PAPS by reverse-phase TLC (see inset), and radiolabeled reactants were quantitated by two-dimensional imaging using a STORM system (Nikon Instruments). Each point is the average of two independent determinations. The solid line through the data is not a statistical fit; rather, it is the behavior predicted using the rate constants in Table 1 and the model in Fig. 5. [P]/[E] is the concentration of product divided by the concentration of enzyme.

FIGURE 3.

Progress curves and simulations of DHEAS formation. The initial reaction conditions were as follows: DHEA (1.1 μm, 1.0 × K_d [E]), DHEAS (0.50 mm, 1.0 × K_d [E]), [³⁵S]PAPS (1.0 μm, 3.7 × K_d [E]), PAP (10 μm, 27 × K_d [E]), MgCl₂ (5.0 mm), and NaPO₄ (50 mm), pH 7.2, T = 25 ± 2 °C. The reaction was allowed to reach what appeared to be equilibrium (7.5 h), at which point the DHEA concentration was increased to 0.50 μm and the reaction was again allowed to plateau. The rate constant for conversion of the product to the substrate central complex was obtained by fitting the data using the 21 other constants (Table 1) associated with the mechanism (Fig. 5). Both phases yielded the same rate constant, 6 (± 0.8) × 10⁻³ s⁻¹, and overall equilibrium constant, 3.8 (± 0.7) × 10⁴.

FIGURE 4.

The mechanism of SULT2A1-catalyzed DHEA sulfation.

FIGURE 5.

Partial substrate inhibition by DHEA. The initial rate of product formation was determined as a function of DHEA concentration. The reaction conditions were as follows: SULT2A1 (0.50 nm), [³H]DHEA (0.30–25 μm), PAPS (30 μm, 100 × K_d [E·DHEA]), MgCl₂ (5.0 mm), and NaPO₄ (50 mm), pH 7.2, T = 25 ± 2 °C. Reactions were initiated by the addition of enzyme. Reactions were quenched with (0.20 m, final). [³H]DHEA was extracted with chloroform, and [³H]DHEAS in the aqueous phase was quantified. Data are plotted as k_cat^app versus [DHEA], where k_cat^app = initial rate/[enzyme active sites]. Each point is the average of two independent determinations. The solid line through the data does not represent a fit of the data; rather, it is the behavior predicted using the 22 constants listed in Table 1 and the model shown in Fig. 5.