Investigations of the catalytic mechanism of thioredoxin glutathione reductase from Schistosoma mansoni.
Investigations of the catalytic mechanism of thioredoxin glutathione reductase from Schistosoma mansoni.
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DOI：
10.1021/bi200107n
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发表时间：
2011-07-05
期刊：
影响因子：
2.9
通讯作者：
Williams DL
中科院分区：
文献类型：
--
作者：
Huang HH;Day L;Cass CL;Ballou DP;Williams CH Jr;Williams DL
曼氏血吸虫的硫氧还蛋白谷胱甘肽还原酶 (SmTGR) 催化硫氧还蛋白和谷胱甘肽二硫化物 (GSSG) 的还原，因此在维持寄生虫的氧化还原稳态中发挥着至关重要的作用。与这一作用相一致，之前的研究表明，SmTGR 是一个有前途的血吸虫病药物靶点。为了帮助开发针对 SmTGR 的有效药物，有必要了解 SmTGR 的催化机制。 SmTGR 是谷胱甘肽还原酶家族中的一种二聚体黄素蛋白，其单体呈头尾排列；每个亚基都包含硫氧还蛋白还原酶 (TrxR) 结构域和谷氧还蛋白 (Grx) 结构域。然而，TrxR 结构域的活性位点由两个亚基的残基组成：一个亚基的 FAD 和氧化还原活性 Cys-154/Cys-159 对，另一个亚基的氧化还原活性 Cys-596'/Sec-597' 对； Grx 结构域的活性位点包含氧化还原活性 Cys-28/Cys-31 对。通过其 Cys-28/Cys-31 二硫醇和/或其 Cys-596'/Sec-597' 硫醇硒酸盐，SmTGR 可以催化 NADPH 还原多种底物。据推测，SmTGR 通过 Cys-28/Cys-31 二硫醇催化去谷胱甘肽反应。我们的厌氧滴定数据表明，NADPH 的还原当量确实可以到达 Grx 结构域中的 Cys-28/Cys-31 二硫化物，以促进该半胱氨酸对实现的还原。为了阐明每个氧化还原活性残基在其各种反应性方面的具体化学作用，我们生成了 SmTGR 的变体。 Cys-28 变体没有 Grx 谷胱甘肽化活性，而 Cys-31 变体保留部分 Grx 谷胱甘肽化活性，表明 Cys-28 硫醇盐是引发去谷胱甘肽化的亲核试剂。当野生型 (WT) SmTGR 在高浓度 GSSG 下孵育时发现的稳态动力学滞后在 Grx 变体中不存在，表明该半胱氨酸对在某种程度上造成了滞后。 Sec-597 变体仍然能够减少多种底物，尽管速度很慢，这表明硒代半胱氨酸很重要，但并不是酶广泛底物耐受性的唯一决定因素。我们的数据表明 Cys-520 和 Cys-574 不太可能参与催化机制。
Thioredoxin glutathione reductase from Schistosoma mansoni (SmTGR) catalyzes the reduction of both thioredoxin and glutathione disulfides (GSSG), thus playing a crucial role in maintaining redox homeostasis in the parasite. In line with this role, previous studies have demonstrated that SmTGR is a promising drug target for schistosomiasis. To aid in the development of efficacious drugs that target SmTGR, it is essential to understand the catalytic mechanism of SmTGR. SmTGR is a dimeric flavoprotein in the glutathione reductase family and it has a head-to-tail arrangement of its monomers; each subunit has the components of both a thioredoxin reductase (TrxR) domain and a glutaredoxin (Grx) domain. However, the active site of the TrxR domain is composed of residues from both subunits: FAD and a redox-active Cys-154/Cys-159 pair from one subunit and a redox-active Cys-596′/Sec-597′ pair from the other; the active site of the Grx domain contains a redox-active Cys-28/Cys-31 pair. Via its Cys-28/Cys-31 dithiol and/or its Cys-596′/Sec-597′ thiol-selenolate, SmTGR can catalyze the reduction of a variety of substrates by NADPH. It is presumed that SmTGR catalyzes deglutathionylation reactions via the Cys-28/Cys-31 dithiol. Our anaerobic titration data suggest that reducing equivalents from NADPH can indeed reach the Cys-28/Cys-31 disulfide in the Grx domain to facilitate reductions effected by this cysteine pair. To clarify the specific chemical roles of each redox-active residue with respect to its various reactivities, we generated variants of SmTGR. Cys-28 variants had no Grx glutathionylation activity whereas Cys-31 variants retained partial Grx glutathionylation activity, indicating that the Cys-28 thiolate is the nucleophile initiating deglutathionylation. Lags in the steady-state kinetics, found when wild-type (WT) SmTGR was incubated at high concentrations of GSSG, were not present in Grx variants, indicating that this cysteine pair is in some way responsible for the lags. A Sec-597 variant was still able to reduce a variety of substrates, albeit slowly, showing that selenocysteine is important but is not the sole determinant for the broad substrate tolerance of the enzyme. Our data show that Cys-520 and Cys-574 are not likely to be involved in the catalytic mechanism.
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影响因子：
15.8
作者：
Abdulla MH;Lim KC;Sajid M;McKerrow JH;Caffrey CR
通讯作者：
Caffrey CR
DOI：
10.1073/pnas.2134510100
发表时间：
2003-10-28
影响因子：
11.1
作者：
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通讯作者：
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影响因子：
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作者：
Discola, Karen Fulan;de Oliveira, Marcos Antonio;Soares Netto, Luis Eduardo
通讯作者：
Soares Netto, Luis Eduardo
影响因子：
4.8
作者：
Gromer, S;Arscott, LD;Becker, K
通讯作者：
Becker, K
影响因子：
4.8
作者：
Bonilla, Mariana;Denicola, Ana;Salinas, Gustavo
通讯作者：
Salinas, Gustavo