Interdisciplinary Graduate Program in Human Toxicology, University of Iowa , Iowa City, Iowa 52242, United States.
Chem Res Toxicol. 2013 Oct 21;26(10):1474-85. doi: 10.1021/tx400207q. Epub 2013 Oct 4.
Human hydroxysteroid sulfotransferase (hSULT2A1) catalyzes the sulfation of a broad range of environmental chemicals, drugs, and other xenobiotics in addition to endogenous compounds that include hydroxysteroids and bile acids. Polychlorinated biphenyls (PCBs) are persistent environmental contaminants, and oxidized metabolites of PCBs may play significant roles in the etiology of their adverse health effects. Quinones derived from the oxidative metabolism of PCBs (PCB-quinones) react with nucleophilic sites in proteins and also undergo redox cycling to generate reactive oxygen species. This, along with the sensitivity of hSULT2A1 to oxidative modification at cysteine residues, led us to hypothesize that electrophilic PCB-quinones react with hSULT2A1 to alter its catalytic function. Thus, we examined the effects of four phenylbenzoquinones on the ability of hSULT2A1 to catalyze the sulfation of the endogenous substrate, dehydroepiandrosterone (DHEA). The quinones studied were 2'-chlorophenyl-2,5-benzoquinone (2'-Cl-BQ), 4'-chlorophenyl-2,5-benzoquinone (4'-Cl-BQ), 4'-chlorophenyl-3,6-dichloro-2,5-benzoquinone (3,6,4'-triCl-BQ), and phenyl-2,5-benzoquinone (PBQ). At all concentrations examined, pretreatment of hSULT2A1 with the PCB-quinones decreased the catalytic activity of hSULT2A1. Pretreatment with low concentrations of PBQ, however, increased the catalytic activity of the enzyme, while higher concentrations inhibited catalysis. A decrease in substrate inhibition with DHEA was seen following preincubation of hSULT2A1 with all of the quinones. Proteolytic digestion of the enzyme followed by LC/MS analysis indicated PCB-quinone- and PBQ-adducts at Cys55 and Cys199, as well as oxidation products at methionines in the protein. Equilibrium binding experiments and molecular modeling suggested that changes due to these modifications may affect the nucleotide binding site and the entrance to the sulfuryl acceptor binding site of hSULT2A1.
人源羟甾类磺基转移酶（hSULT2A1）除了催化内源性化合物（如羟甾类和胆酸）的磺化作用外，还能催化广泛的环境化学物质、药物和其他外源性物质的磺化作用。多氯联苯（PCBs）是持久性环境污染物，PCBs 的氧化代谢产物可能在其不良健康影响的病因中发挥重要作用。PCBs 氧化代谢物（PCB-醌）与蛋白质中的亲核位点反应，并经历氧化还原循环产生活性氧物种。这一点，加上 hSULT2A1 对半胱氨酸残基氧化修饰的敏感性，使我们假设亲电 PCB-醌与 hSULT2A1 反应，改变其催化功能。因此，我们研究了四种苯并醌对 hSULT2A1 催化内源性底物脱氢表雄酮（DHEA）磺化能力的影响。研究的醌是 2'-氯苯基-2,5-苯醌（2'-Cl-BQ）、4'-氯苯基-2,5-苯醌（4'-Cl-BQ）、4'-氯苯基-3,6-二氯-2,5-苯醌（3,6,4'-三氯-BQ）和苯并醌（PBQ）。在所有研究的浓度下，hSULT2A1 与 PCB-醌预处理都会降低 hSULT2A1 的催化活性。然而，低浓度 PBQ 的预处理会增加酶的催化活性，而较高浓度则会抑制催化作用。与所有醌预孵育后，观察到 DHEA 对底物的抑制作用降低。用 LC/MS 分析对酶进行蛋白水解消化表明，在 Cys55 和 Cys199 处有 PCB-醌和 PBQ 加合物，以及蛋白质中蛋氨酸的氧化产物。平衡结合实验和分子建模表明，这些修饰引起的变化可能影响 hSULT2A1 的核苷酸结合位点和硫酸根供体结合位点的入口。
