Modification of the Catalytic Function of Human Hydroxysteroid Sulfotransferase hSULT2A1 by Formation of Disulfide Bonds

Abstract

The human cytosolic sulfotransferase hSULT2A1 catalyzes the sulfation of a broad range of xenobiotics, as well as endogenous hydroxysteroids and bile acids. Reversible modulation of the catalytic activity of this enzyme could play important roles in its physiologic functions. Whereas other mammalian sulfotransferases are known to be reversibly altered by changes in their redox environment, this has not been previously shown for hSULT2A1. We have examined the hypothesis that the formation of disulfide bonds in hSULT2A1 can reversibly regulate the catalytic function of the enzyme. Three thiol oxidants were used as model compounds to investigate their effects on homogeneous preparations of hSULT2A1: glutathione disulfide, 5,59-dithiobis(2-nitrobenzoic acid), and 1,1'-azobis(N,N-dimethylformamide) (diamide). Examination of the effects of disulfide bond formation with these agents indicated that the activity of the enzyme is reversibly altered. Studies on the kinetics of the hSULT2A1-catalyzed sulfation of dehydroepiandrosterone (DHEA) showed the effects of disulfide bond formation on the substrate inhibition characteristics of the enzyme. The effects of these agents on the binding of substrates and products, liquid chromatography-mass spectrometry identification of the disulfides formed, and structural modeling of the modified enzyme were examined. Our results indicate that conformational changes at cysteines near the nucleotide binding site affect the binding of both the nucleotide and DHEA to the enzyme, with the specific effects dependent on the structure of the resulting disulfide. Thus, the formation of disulfide bonds in hSULT2A1 is a potentially important reversible mechanism for alterations in the rates of sulfation of both endogenous and xenobiotic substrates.

Chlorinated Biphenyl Quinones and Phenyl-2,5-benzoquinone Differentially Modify the Catalytic Activity of Human Hydroxysteroid Sulfotransferase hSULT2A1

“…41 Indeed, our current results on the modification of hSULT2A1 with three PCB-quinones also showed consistent decreases in catalytic activity. There was, however, an increase in the catalytic activity at some concentrations of the non-chlorinated biphenyl quinone, PBQ.…”

“…41,55,57,65–68 While most of these SULTs have been members of family 1, we have recently reported that the catalytic activity of human hydroxysteroid sulfotransferase hSULT2A1 is also sensitive to formation of disulfide bonds at key cysteine residues. 41 Alteration of the structure and function of hSULT2A1 through oxidative post-translational changes at cysteine residues led us to hypothesize that changes in catalytic function may also result from reaction of the enzyme with electrophiles that form adducts at those cysteine residues. Such adduct formation would likely alter sulfation with concomitant effects on the metabolism of xenobiotics as well as endogenous molecules.…”

“…41 Removal of DTT to a concentration less than 0.01 mM was then verified by a standard assay for determination of thiols. 51 Following the removal of DTT, hSULT2A1 was incubated for 1 hour at 25 °C with the indicated concentrations of PCB-quinones or PBQ in the same buffer solution that had been used for chromatography on the PD-10 column ( i.e ., 50 mM Tris-HCl buffer, pH 7.4, containing 0.25 M sucrose, 10 % (v/v) glycerol, and 0.05 % (v/v) Tween 20).…”

Cytochrome c adducts with PCB quinoid metabolites

“…One possibility is that the reduction may be caused by PCB3 hydroquinone or semiquinone radicals, as described with benzoquinones (Williams 1963, Winterbourn 1981). Interestingly, after adduct formation with sulfhydryl groups in peptides/proteins like N-acetyl cysteine (Wangpradit et al 2009) and human hydroxysteroid sulfotransferase hSULT2A1 (Qin et al 2013b) only hydroquinone derivatives of PCBs were identified, while the adducts we found with cytochrome c were all quinoid derivatives of PCBs. This may be caused by the oxidation of a hydroquinone or semiquinone adduct by the ferric heme group in cytochrome c resulting in reduced cytochrome c and a PCB quinone adduct.…”

Toxicological and therapeutic implications of interactions between polychlorinated biphenyl sulfates and human transthyretin

“…60,82 PCB quinones are also capable of protein adduct formation, a mechanism by which they can interfere with the physiological functions of a protein. [83][84][85] OHPCBs also represent substrates for conjugation reactions catalyzed by sulfotransferases (SULTs), UDP-glucuronosyl transferases (UGTs) or glutathione Stransferases (GSTs) to yield their respective sulfate, glucuronic acid or mercapturic acid conjugates. 73,86,87 These conjugation reactions increase the polarity of PCBs, which results in enhanced hydrophilicity and which is typically assumed to result in renal or biliary excretion.…”