BACKGROUND We previously showed that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is S-glutathionylated in the presence of H2O2 and GSH. S-glutathionylation was shown to result in the formation of a disulfide bridge in the active site of the protein. In the present work, the possible biological significance of the disulfide bridge was investigated. METHODS Human recombinant GAPDH with the mutation C156S (hGAPDH_C156S) was obtained to prevent the formation of the disulfide bridge. Properties of S-glutathionylated hGAPDH_C156S were studied in comparison with those of the wild-type protein hGAPDH. RESULTS S-glutathionylation of hGAPDH and hGAPDH_C156S results in the reversible inactivation of the proteins. In both cases, the modification results in corresponding mixed disulfides between the catalytic Cys152 and GSH. In the case of hGAPDH, the mixed disulfide breaks down yielding Cys152-Cys156 disulfide bridge in the active site. In hGAPDH_C156S, the mixed disulfide is stable. Differential scanning calorimetry method showed that S-glutathionylation leads to destabilization of hGAPDH molecule, but does not affect significantly hGAPDH_C156S. Reactivation of S-glutathionylated hGAPDH in the presence of GSH and glutaredoxin 1 is approximately two-fold more efficient compared to that of hGAPDH_C156S. CONCLUSIONS S-glutathionylation induces the formation of Cys152-Cys156 disulfide bond in the active site of hGAPDH, which results in structural changes of the protein molecule. Cys156 is important for reactivation of S-glutathionylated GAPDH by glutaredoxin 1. GENERAL SIGNIFICANCE The described mechanism may be important for interaction between GAPDH and other proteins and ligands, involved in cell signaling.

中文翻译：

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人甘油3-磷酸甘油醛脱氢酶的S-谷胱甘肽化和Cys152-Cys156二硫键在蛋白质活性位点中的可能作用。

背景技术我们先前显示，在H2O2和GSH存在下，甘油醛-3-磷酸脱氢酶（GAPDH）被S-谷胱甘肽化。已显示S-谷胱甘肽酰化导致在蛋白质的活性位点形成二硫键。在目前的工作中，研究了二硫键的可能的生物学意义。方法获得具有C156S突变的人重组GAPDH（hGAPDH_C156S），以防止二硫键的形成。与野生型蛋白hGAPDH的性质相比，研究了S-谷胱甘肽化的hGAPDH_C156S的性质。结果hGAPDH和hGAPDH_C156S的S-谷胱甘肽酰化导致蛋白质的可逆失活。在两种情况下，修饰都会在催化Cys152和GSH之间产生相应的混合二硫化物。对于hGAPDH，混合的二硫键分解，在活性位点产生Cys152-Cys156二硫键。在hGAPDH_C156S中，混合的二硫化物是稳定的。差示扫描量热法显示S-谷胱甘肽化导致hGAPDH分子不稳定，但对hGAPDH_C156S没有明显影响。与hGAPDH_C156S相比，在GSH和谷胱甘肽毒素1的存在下S-谷胱甘肽化的hGAPDH的重新激活效率大约高两倍。结论S-谷胱甘肽酰化诱导hGAPDH活性位点中Cys152-Cys156二硫键的形成，导致蛋白质分子的结构变化。Cys156对戊二醛1激活S-谷胱甘肽化的GAPDH很重要。一般意义所述机制对于GAPDH与其他蛋白质和配体之间的相互作用可能很重要，