3-磷酸甘油醛脱氢酶的S-谷胱甘肽酰化诱导在该酶的活性位点形成C150-C154内亚基二硫键。,Biochimica et Biophysica Acta (BBA) - General Subjects

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3-磷酸甘油醛脱氢酶的S-谷胱甘肽酰化诱导在该酶的活性位点形成C150-C154内亚基二硫键。
Biochimica et Biophysica Acta (BBA) - General Subjects ( IF 2.8 ) Pub Date : 2017-09-25 , DOI: 10.1016/j.bbagen.2017.09.008
K V Barinova ₁ , M V Serebryakova ₂ , V I Muronetz ₁ , E V Schmalhausen ₂

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背景技术3-磷酸甘油醛脱氢酶（GAPDH）是一种参与许多非糖酵解功能的糖酵解蛋白。在植物和动物组织中发现了S-谷胱甘肽化的GAPDH。GAPDH S-谷胱甘肽酰化的作用尚不完全清楚。方法在过氧化氢和还原型谷胱甘肽（GSH）存在下，兔肌肉GAPDH被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法测定修饰的蛋白质。结果在H2O2和GSH共同作用下，GAPDH的孵育导致了该酶的完全失活。与过氧化氢不可逆地氧化GAPDH相比，这种修饰可以在过量的GSH或二硫苏糖醇中逆转。根据MALDI-MS分析数据，修饰的蛋白质既包含Cys150和GSH之间的混合二硫键，又包含Cys150和Cys154之间的亚基内部二硫键（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的三级结构松散，降低了其对NAD +的亲和力和热稳定性。结论Cys150和GSH之间的混合二硫键是S-谷胱甘肽酰化的中间产物：其随后与Cys154的反应在GAPDH的活性位点产生亚单位内二硫键。混合的二硫键和C150-C154二硫键可保护GAPDH免受不可逆的氧化，并可以在过量的硫醇中被还原。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，提示S-谷胱甘肽化在氧化还原信号传导中的作用。一般意义该手稿考虑了氧化还原调节细胞功能的可能机制之一。

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

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 H2O2 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 H2O2 together with GSH resulted in the complete inactivation of the enzyme. In contrast to irreversible oxidation of GAPDH by H2O2, 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.

更新日期：2019-11-01

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