Mechanism of Thiol-Supported Arsenate Reduction Mediated by Phosphorolytic-Arsenolytic Enzymes
Mechanism of Thiol-Supported Arsenate Reduction Mediated by Phosphorolytic-Arsenolytic Enzymes
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DOI：
10.1093/toxsci/kfp113
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发表时间：
2009-08-01
影响因子：
3.8
通讯作者：
Nemeti, Balazs
中科院分区：
文献类型：
--
作者：
Gregus, Zoltan;Roos, Goedele;Nemeti, Balazs
催化其底物磷酸化裂解的酶可以在还原剂如谷胱甘肽或二硫代treitol （DTT）的存在下，通过裂解砷酸盐（AsV）还原为毒性更强的亚砷酸盐（AsIII）。我们已经在嘌呤核苷磷酸化酶（PNP）、甘油醛-3-磷酸脱氢酶（GAPDH）、糖原磷酸化酶-a （GPa）和磷酸转乙酰化酶（PTA）中证明了这一点。采用多学科方法，我们探索了这些酶介导AsV减少的机制。已知PNP将带AsV的肌苷裂解为次黄嘌呤和核糖-1-砷酸盐。在肌苷、AsV和DTT存在下，PNP介导AsIII的形成。在本研究中，我们先用肌苷和AsV孵育PNP，使其进行溶砷反应，然后用PNP抑制剂BCX-1777阻断该反应，加入DTT继续孵育。尽管PNP有抑制作用，但在这些孵育中仍形成了大量的AsIII，这表明PNP并不直接降低AsV，而是形成一种产物（即核糖-1-砷酸盐），该产物通过DTT还原为AsIII。对其他溶砷酶（GPa、GAPDH和PTA）的类似研究也得出了类似的结果。在pnp催化的砷分解过程中（DTT类似于二巯基丙烷-1-磺酸>巯基乙醇> DMSA > GSH），不同的硫醇在pnp催化的短暂的砷分解过程中加入时，同样支持AsV的还原，形成核糖-1-砷酸盐。BCX-1777后逐渐延迟添加DTT的实验表明，核糖-1-砷酸盐的半衰期很短，为4分钟。综上所述，磷酸解酶，如PNP、GAPDH、GPa和PTA，促进了硫醇依赖性AsV的还原，因为它们比AsV更容易将AsV转化为硫醇可还原的砷化产物。为了支持这一观点，使用概念密度泛函理论的反应性描述符（局部柔软性，核核性）的反应性研究表明，硫醇对砷化代谢物的还原比AsV更有利。
Enzymes catalyzing the phosphorolytic cleavage of their substrates can reduce arsenate (AsV) to the more toxic arsenite (AsIII) via the arsenolytic substrate cleavage in presence of a reductant, as glutathione or dithiotreitol (DTT). We have shown this for purine nucleoside phosphorylase (PNP), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glycogen phosphorylase-a (GPa), and phosphotransacetylase (PTA). Using a multidisciplinary approach, we explored the mechanism whereby these enzymes mediate AsV reduction. It is known that PNP cleaves inosine with AsV into hypoxanthine and ribose-1-arsenate. In presence of inosine, AsV and DTT, PNP mediates AsIII formation. In this study, we incubated PNP first with inosine and AsV, allowing the arsenolytic reaction to run, then blocked this reaction with the PNP inhibitor BCX-1777, added DTT and continued the incubation. Despite inhibition of PNP, large amount of AsIII was formed in these incubations, indicating that PNP does not reduce AsV directly but forms a product (i.e., ribose-1-arsenate) that is reduced to AsIII by DTT. Similar studies with the other arsenolytic enzymes (GPa, GAPDH, and PTA) yielded similar results. Various thiols that differentially supported AsV reduction when present during PNP-catalyzed arsenolysis (DTT similar to dimercaptopropane-1-sulfonic acid > mercaptoethanol > DMSA > GSH) similarly supported AsV reduction when added only after a transient PNP-catalyzed arsenolysis, which preformed ribose-1-arsenate. Experiments with progressively delayed addition of DTT after BCX-1777 indicated that ribose-1-arsenate is short-lived with a half-life of 4 min. In conclusion, phosphorolytic enzymes, such as PNP, GAPDH, GPa, and PTA, promote thiol-dependent AsV reduction because they convert AsV into arsenylated products reducible by thiols more readily than AsV. In support of this view, reactivity studies using conceptual density functional theory reactivity descriptors (local softness, nucleofugality) indicate that reduction by thiols of the arsenylated metabolites is favored over AsV.