Faculty of Medicine, Institute of Biochemistry, University of Leipzig, Leipzig, Germany.
PLoS One. 2010 Apr 29;5(4):e10399. doi: 10.1371/journal.pone.0010399.
Glyoxalase 1 (Glo1) and glyoxalase 2 (Glo2) are ubiquitously expressed cytosolic enzymes that catalyze the conversion of toxic alpha-oxo-aldehydes into the corresponding alpha-hydroxy acids using L-glutathione (GSH) as a cofactor. Human Glo1 exists in various isoforms; however, the nature of its modifications and their distinct functional assignment is mostly unknown.
METHODOLOGY/PRINCIPAL FINDINGS: We characterized native Glo1 purified from human erythrocytes by mass spectrometry. The enzyme was found to undergo four so far unidentified posttranslational modifications: (i) removal of the N-terminal methionine 1, (ii) N-terminal acetylation at alanine 2, (iii) a vicinal disulfide bridge between cysteine residues 19 and 20, and (iv) a mixed disulfide with glutathione on cysteine 139. Glutathionylation of Glo1 was confirmed by immunological methods. Both, N-acetylation and the oxidation state of Cys(19/20), did not impact enzyme activity. In contrast, glutathionylation strongly inhibited Glo1 activity in vitro. The discussed mechanism for enzyme inhibition by glutathionylation was validated by molecular dynamics simulation.
CONCLUSION/SIGNIFICANCE: It is shown for the first time that Glo1 activity directly can be regulated by an oxidative posttranslational modification that was found in the native enzyme, i.e., glutathionylation. Inhibition of Glo1 by chemical reaction with its co-factor and the role of its intramolecular disulfides are expected to be important factors within the context of redox-dependent regulation of glucose metabolism in cells.
醛糖还原酶 1（Glo1）和醛糖还原酶 2（Glo2）是广泛表达的细胞溶质酶，它们使用 L-谷胱甘肽（GSH）作为辅因子催化有毒的α-氧代醛转化为相应的α-羟基酸。人 Glo1 存在多种同工型；然而，其修饰的性质及其独特的功能分配在很大程度上尚不清楚。
方法/主要发现：我们通过质谱法对从人红细胞中纯化的天然 Glo1 进行了表征。该酶被发现经历了四种迄今尚未确定的翻译后修饰：（i）去除 N 端甲硫氨酸 1，（ii）N 端丙氨酸 2 的乙酰化，（iii）半胱氨酸残基 19 和 20 之间的毗邻二硫键，以及（iv）与半胱氨酸 139 上的谷胱甘肽的混合二硫键。免疫方法证实了 Glo1 的谷胱甘肽化。N-乙酰化和 Cys（19/20）的氧化状态都没有影响酶活性。相比之下，谷胱甘肽化在体外强烈抑制 Glo1 活性。通过分子动力学模拟验证了讨论的酶抑制机制。
结论/意义：首次表明 Glo1 活性可直接受到氧化翻译后修饰的调节，该修饰在天然酶中发现，即谷胱甘肽化。化学与酶的辅因子反应抑制 Glo1 以及其分子内二硫键的作用有望成为细胞内葡萄糖代谢的氧化还原依赖性调节的重要因素。
