Glyceraldehyde-3-phosphate dehydrogenase 的 S-谷胱甘肽化诱导酶活性部位的 C150-C154 亚基内二硫键的形成。

S-glutathionylation of glyceraldehyde-3-phosphate dehydrogenase induces formation of C150-C154 intrasubunit disulfide bond in the active site of the enzyme.

机构信息

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119234, Russia; Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow 119234, Russia.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119234, Russia.

出版信息

Biochim Biophys Acta Gen Subj. 2017 Dec;1861(12):3167-3177. doi: 10.1016/j.bbagen.2017.09.008. Epub 2017 Sep 19.

DOI:10.1016/j.bbagen.2017.09.008

PMID:28935607

Abstract

BACKGROUND

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a glycolytic protein involved in numerous non-glycolytic functions. S-glutathionylated GAPDH was revealed in plant and animal tissues. The role of GAPDH S-glutathionylation is not fully understood.

METHODS

Rabbit muscle GAPDH was S-glutathionylated in the presence of HO and reduced glutathione (GSH). The modified protein was assayed by MALDI-MS analysis, differential scanning calorimetry, dynamic light scattering, and ultracentrifugation.

RESULTS

Incubation of GAPDH in the presence of HO together with GSH resulted in the complete inactivation of the enzyme. In contrast to irreversible oxidation of GAPDH by HO, this modification could be reversed in the excess of GSH or dithiothreitol. By data of MALDI-MS analysis, the modified protein contained both mixed disulfide between Cys150 and GSH and the intrasubunit disulfide bond between Cys150 and Cys154 (different subunits of tetrameric GAPDH may contain different products). S-glutathionylation results in loosening of the tertiary structure of GAPDH, decreases its affinity to NAD and thermal stability.

CONCLUSIONS

The mixed disulfide between Cys150 and GSH is an intermediate product of S-glutathionylation: its subsequent reaction with Cys154 results in the intrasubunit disulfide bond in the active site of GAPDH. The mixed disulfide and the C150-C154 disulfide bond protect GAPDH from irreversible oxidation and can be reduced in the excess of thiols. Conformational changes that were observed in S-glutathionylated GAPDH may affect interactions between GAPDH and other proteins (ligands), suggesting the role of S-glutathionylation in the redox signaling.

GENERAL SIGNIFICANCE

The manuscript considers one of the possible mechanisms of redox regulation of cell functions.

摘要

背景

甘油醛-3-磷酸脱氢酶（GAPDH）是一种参与多种非糖酵解功能的糖酵解蛋白。已在植物和动物组织中发现 S-谷胱甘肽化的 GAPDH。GAPDH S-谷胱甘肽化的作用尚未完全了解。

方法

在 HO 和还原型谷胱甘肽 (GSH) 的存在下，兔肌肉 GAPDH 发生 S-谷胱甘肽化。通过 MALDI-MS 分析、差示扫描量热法、动态光散射和超速离心测定修饰蛋白。

结果

在 HO 和 GSH 存在下孵育 GAPDH 导致酶完全失活。与 HO 对 GAPDH 的不可逆氧化相反，这种修饰可以在 GSH 或二硫苏糖醇过量时逆转。根据 MALDI-MS 分析的数据，修饰蛋白同时含有 Cys150 与 GSH 之间的混合二硫键和 Cys150 与 Cys154 之间的亚基内二硫键（四聚体 GAPDH 的不同亚基可能包含不同的产物）。S-谷胱甘肽化导致 GAPDH 三级结构松弛，降低其与 NAD 的亲和力和热稳定性。

结论

Cys150 与 GSH 之间的混合二硫键是 S-谷胱甘肽化的中间产物：其随后与 Cys154 的反应导致 GAPDH 活性位点中的亚基内二硫键。混合二硫键和 C150-C154 二硫键保护 GAPDH 免受不可逆氧化，并可在硫醇过量时还原。在 S-谷胱甘肽化的 GAPDH 中观察到的构象变化可能会影响 GAPDH 与其他蛋白质（配体）之间的相互作用，表明 S-谷胱甘肽化在氧化还原信号转导中的作用。