Glutathionylation of cytosolic glyceraldehyde-3-phosphate dehydrogenase from the model plant Arabidopsis thaliana is reversed by both glutaredoxins and thioredoxins in vitro
Glutathionylation of cytosolic glyceraldehyde-3-phosphate dehydrogenase from the model plant Arabidopsis thaliana is reversed by both glutaredoxins and thioredoxins in vitro
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DOI：
10.1042/bj20120505
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发表时间：
2012-08-01
影响因子：
4.1
通讯作者：
Trost, Paolo
中科院分区：
文献类型：
--
作者：
Bedhomme, Mariette;Adamo, Mattia;Trost, Paolo
植物含有细胞质和叶绿体GAPDH（甘油醛-3-磷酸脱氢酶）。在拟南芥中，胞质GAPDH参与糖酵解途径，并由两种差异表达的同种型（GapC 1和GapC 2）代表，其氨基酸序列具有98%的相同性。在本研究中，我们发现GapC 1是一种磷酸化NAD特异性GAPDH，其酶活性严格依赖于Cyst（149）。催化Cys（149）是蛋白质中唯一暴露于溶剂的胱氨酸，其巯基相对酸性（pK（a）= 5.7）。该性质使得GapCl对H2 O2的氧化敏感，H2 O2似乎通过将Cys（149）的硫醇盐（-S-）经由不稳定的次磺酸盐中间体（-SO-）转化为不可逆的氧化形式（-SO2-和-SO 3-）来抑制酶活性。GSH（还原型谷胱甘肽）通过与Cys（149）亚磺酸盐反应产生混合二硫化物（Cys（149）-SSG）来防止这一不可逆过程，如MS和生物素化GSH所示。谷胱甘肽化的GapC 1可以通过GSH依赖性单硫醇机制被胞质谷氧还蛋白完全再激活，或者通过NADPH：硫氧还蛋白还原酶生理还原的胞质硫氧还蛋白不太有效地被再激活。根据GAPDH在真核细胞中的多种功能（例如糖酵解、基因表达控制和细胞凋亡）讨论了这些发现的潜在相关性，这些功能似乎受到催化半胱氨酸氧化还原状态的影响（149）。
Plants contain both cytosolic and chloroplastic GAPDHs (glyceraldehyde-3-phosphate dehydrogenases). In Arabidopsis thaliann, cytosolic GAPDH is involved in the glycolytic pathway and is represented by two differentially expressed isoforms (GapC1 and GapC2) that are 98% identical in amino acid sequence. In the present study we show that GapC1 is a phosphorylating NAD-specific GAPDH with enzymatic activity strictly dependent on Cyst(149). Catalytic Cys(149) is the only solvent-exposed cystine of the protein and its thiol is relatively acidic (pK(a) = 5.7). This property makes GapCl sensitive to oxidation by H2O2, which appears to inhibit enzyme activity by converting the thiolate of Cys(149) (-S-) into irreversible oxidized forms (-SO2- and -SO3-) via a labile sulfenate intermediate (-SO-). GSH (reduced glutathione) prevents this irreversible process by reacting with Cys(149) sulfenates to give rise to a mixed disulfide (Cys(149)-SSG), as demonstrated by both MS and biotinylated GSH. Glutathionylated GapC1 can be fully reactivated either by cytosolic glutaredoxin, via a GSHdependent monothiol mechanism, or, less efficiently, by cytosolic thioredoxins physiologically reduced by NADPH:thioredoxin reductase. The potential relevance of these findings is discussed in the light of the multiple functions of GAPDH in eukaryotic cells (e.g. glycolysis, control of gene expression and apoptosis) that appear to be influenced by the redox state of the catalytic cys(149).