Mechanistic studies of β-arylsulfotransferase IV

doi:10.1073/pnas.0337638100

Mechanistic studies of β-arylsulfotransferase IV

Department of Chemistry and Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037

Contributed by Chi-Huey Wong

Abstract

Sulfotransferases are an important class of enzymes that catalyze the transfer of a sulfuryl group to a hydroxyl or amine moiety on various molecules including small-molecule drugs, steroids, hormones, carbohydrates, and proteins. They have been implicated in a number of disease states but remain poorly understood, complicating the design of specific, small-molecule inhibitors. A linear free-energy analysis in both the forward and reverse directions indicates that the transfer of a sulfuryl group to an aryl hydroxyl group catalyzed by β-arylsulfotransferase IV likely proceeds by a dissociative (sulfotrioxide-like) mechanism. Values for the Brønsted coefficients (β_nuc and β_lg) are +0.33 and −0.45, giving Leffler α values of 0.19 and 0.61 for the forward and reverse reactions, respectively.

sulfotransferase‖linear free energy‖mechanism

Footnotes

↵ * To whom correspondence should be addressed. E-mail: wong{at}scripps.edu.
↵ † The K _m of PAPS was measured at pH 6.0 (41 ± 3 mM) to assure PAPS was saturating at all pH ranges used. All molecules were scanned between 350 and 450 nm. In each case 405 nm was very near the optimum wavelength (within ±3 nm). All extinction coefficients used were measured at 405 nm.
↵ ‡ The β_nuc value for the neutral phenol was calculated at pH 6.2 for PNP, 2-fluoro-4-nitrophenol, and 3-fluoro-4-nitrophenol by using Eq. 1 (41, 42). The data showed a linear relationship with a slope of −0.3. In this case, the 3-fluoro-4-nitrophenol is 89% ionized, the 2-fluoro-4-nitrophenol is 50% ionized, and the PNP is 11% ionized. Further work needs to be done by using less acidic phenols, but this limited data set is further argument for the partially negative species being the nucleophile in the transition state and the active-site histidine perhaps acting as a base to remove the proton in the neutral case.
↵ § For 2,3,5,6-tetrafluoro-4-nitrophenol we noticed that the reaction was inhibited, and thus we measured the inhibition constant (K _i = 0.98mM). This inhibition might be due to the exceptionally poor nucleophilicity of this molecule or a difference in the way the molecule binds in the aryl-binding site. Further experiments must be done to determine this.
Abbreviations:

ST,

sulfotransferase;

PAPS,

3′-phospho-adenosyl-5′-phosphosulfate;

PAP,

3′-phosphoadenosyl-5′-phosphate;

β-AST-IV,

β-arylsulfotransferase IV;

PNP,

p-nitrophenol