Oxidative and electrophilic structural modification and catalytic regulation of human hydroxysteroid sulfotransferase 2a1 (hsult2a1)

Human hydroxysteroid sulfotransferase (hSULT2A1) catalyzes the sulfation of a broad range of endogenous (e.g., hormones, neurotransmitters, bile acids) as well as xenobiotic (e.g, drugs, environmental pollutants) compounds. Alteration in the catalytic activity of hSULT2A1 can lead to outcomes like endocrine disruptions or aberrant drug metabolism and xenobiotic toxicity. Oxidative and electrophilic stresses are known to cause physiological damage and be implicated as possible underlying pathologic mechanisms of a wide range of diseases. To examine the oxidative as well as electrophilic regulation of hSULT2A1, model oxidants (glutathione disulfide (GSSG), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), diamide, tert-butyl hydroperoxide (TBHP)) and electrophiles such as quinone metabolites of polychlorinated biphenyls (PCB-quinones) and phenyl-p- benzoquinone were chosen for this study. Mechanistic studies correlating the enzyme structural modifications with alteration in the catalytic properties were performed to elucidate the catalytic regulative mechanism of an individual oxidant or electrophile.

Thiol oxidants including GSSG, DTNB, and diamide showed catalytic regulation of hSULT2A1. Changes in protein intrinsic fluorescence indicated conformational alterations in hSULT2A1 following the reaction with diamide. Binding properties of hSULT2A1 for its substrates were also altered after reaction with these thiol oxidants, which could be one major reason for the kinetic alteration due to oxidative modification. Formation of mixed disulfides with cysteines in hSULT2A1 was also identified as a result of reaction with GSSG and DTNB.

TBHP was chosen as a model for lipid peroxides, and reaction with this hydroperoxide decreased the catalytic function of hSULT2A1. The dissociation constant for binding of dehydroepiandrosterone (DHEA) was significantly altered with TBHP-pretreatment, but this did not affect the binding of 3',5'-adenosine diphosphate (PAP) to the enzyme. Structural analysis identified cysteine sulfonic acids and methionine sulfoxide formation after reaction of hSULT2A1 with TBHP, which could account for the alterations in the binding properties and the catalytic activity.

Both PCB-quinones and PBQ could regulate the catalytic activity of hSULT2A1. Although PCB-quinones only caused decreases in the catalytic activity at all concentrations tested, pretreatment with PBQ indicated that lower concentrations resulted in an increase in the catalytic activity of hSULT2A1 that was followed by a decrease in the catalytic activity of hSULT2A1 upon increasing the concentration of PBQ in the pretreatment. Differences in the dissociation constants of PAP after PBQ-pretreatment were also observed, indicating the key role played by these PCB-quinones in altering the binding of either PAP or the sulfuryl donors, PAPS. Adducts at cysteines in hSULT2A1 were formed following reactions with PCB-quinones and PBQ. Small amounts of cysteine sulfonic acids and methionine sulfoxides were also formed following reaction of the protein with PCB-quinones and PBQ.

Therefore, alterations in both the catalytic function as well as the structural properties of hSULT2A1 by interaction with oxidants and electrophiles may lead to changes in the metabolism of xenobiotics, as well as alterations in the endogenous balance of various steroid hormones. Such changes may be an important component in physiological damage that occurs under oxidative and electrophilic stress.