A novel mechanism of functional cooperativity regulation by thiol redox status in a dimeric inorganic pyrophosphatase
A novel mechanism of functional cooperativity regulation by thiol redox status in a dimeric inorganic pyrophosphatase
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二聚体无机焦磷酸酶中硫醇氧化还原状态的功能协同调节的新机制
DOI：
10.1016/j.bbagen.2016.09.017
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发表时间：
2017
期刊：
影响因子：
--
通讯作者：
Logullo C.
中科院分区：
文献类型：
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作者：
Costa EP;Facanha AR;Cruz CS;Silva JN;Machado JA;Carvalho GM;Fernandes MR;Martins R;Campos E;Romeiro NC;Githaka NW;Konnai S;Ohashi K;Vaz IS Jr;Logullo C.
背景无机焦磷酸酶是将焦磷酸盐转化为正磷酸盐的重要金属依赖性酶。该反应是相当放能的，并且为许多ATP驱动的生物合成反应提供了热力学优势。我们以前已经证明，胞质PPase从R。microplusembryos是一种非典型的家族I PPase。在这里，我们探讨了位于同型二聚体界面的半胱氨酸残基的功能作用，其氧化还原敏感性，以及与巯基氧化还原状态相关的结构和动力学参数。蜱胚胎和计算方法来分析和预测关键氨基酸以及同二聚体界面的物理化学性质。位于同二聚体界面，被发现在稳定两个催化位点之间的功能协同性中发挥重要作用，如WT-rBmPPase的动力学和希尔系数分析所示。WT-rBmPPase活性被生理抗氧化分子如还原型谷胱甘肽和抗坏血酸上调。另一方面，过氧化氢在生理浓度下降低了WT-rBmPPase对底物（PPi）的亲和力，可能是通过诱导二硫键形成。ConclusionsOur结果提供了一个新的角度，在理解氧化还原控制二硫键形成的酶从吸血节肢动物。下调的可逆性依赖于疏水相互作用在二聚体interfaces.General significanceThis研究是第一次报告的可溶性PPase的二聚体协同调节的氧化还原机制，根据半胱氨酸的氧化还原状态。
BackgroundInorganic PPases are essential metal-dependent enzymes that convert pyrophosphate into orthophosphate. This reaction is quite exergonic and provides a thermodynamic advantage for many ATP-driven biosynthetic reactions. We have previously demonstrated that cytosolic PPase fromR. microplusembryos is an atypical Family I PPase. Here, we explored the functional role of the cysteine residues located at the homodimer interface, its redox sensitivity, as well as structural and kinetic parameters related to thiol redox status.MethodsIn this work, we used prokaryotic expression system for recombinant protein overexpression, biochemical approaches to assess kinetic parameters, ticks embryos and computational approaches to analyze and predict critical amino acids as well as physicochemical properties at the homodimer interface.ResultsCysteine 339, located at the homodimer interface, was found to play an important role in stabilizing a functional cooperativity between the two catalytic sites, as indicated by kinetics and Hill coefficient analyses of the WT-rBmPPase. WT-rBmPPase activity was up-regulated by physiological antioxidant molecules such as reduced glutathione and ascorbic acid. On the other hand, hydrogen peroxide at physiological concentrations decreased the affinity of WT-rBmPPase for its substrate (PPi), probably by inducing disulfide bridge formation.ConclusionsOur results provide a new angle in understanding redox control by disulfide bonds formation in enzymes from hematophagous arthropods. The reversibility of the down-regulation is dependent on hydrophobic interactions at the dimer interface.General significanceThis study is the first report on a soluble PPase where dimeric cooperativity is regulated by a redox mechanism, according to cysteine redox status.