Division of Redox Regulation, DKFZ-ZMBH Alliance, German Cancer Research Center (DKFZ), Heidelberg, Germany.
1] Molecular Biomechanics, Heidelberg Institute of Theoretical Studies, Heidelberg, Germany. [2] Faculty of Chemistry, University of Heidelberg, Heidelberg, Germany.
Nat Chem Biol. 2015 Feb;11(2):156-63. doi: 10.1038/nchembio.1720. Epub 2015 Jan 12.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is sensitive to reversible oxidative inactivation by hydrogen peroxide (H2O2). Here we show that H2O2 reactivity of the active site thiolate (C152) is catalyzed by a previously unrecognized mechanism based on a dedicated proton relay promoting leaving group departure. Disruption of the peroxidatic reaction mechanism does not affect the glycolytic activity of GAPDH. Therefore, specific and separate mechanisms mediate the reactivity of the same thiolate nucleophile toward H2O2 and glyceraldehyde 3-phosphate, respectively. The generation of mutants in which the glycolytic and peroxidatic activities of GAPDH are comprehensively uncoupled allowed for a direct assessment of the physiological relevance of GAPDH H2O2 sensitivity. Using yeast strains in which wild-type GAPDH was replaced with H2O2-insensitive mutants retaining full glycolytic activity, we demonstrate that H2O2 sensitivity of GAPDH is a key component of the cellular adaptive response to increased H2O2 levels.
甘油醛-3-磷酸脱氢酶（GAPDH）对过氧化氢（H2O2）的可逆氧化失活敏感。在这里，我们表明，基于促进离去基团离去的专用质子传递，活性位点巯基（C152）的 H2O2 反应性由以前未被识别的机制催化。过氧化物反应机制的破坏并不影响 GAPDH 的糖酵解活性。因此，针对 H2O2 和甘油醛 3-磷酸，分别介导同一巯基亲核试剂反应性的是特定且独立的机制。生成突变体，其中 GAPDH 的糖酵解和过氧化物酶活性被全面解偶联，允许直接评估 GAPDH 对 H2O2 敏感性的生理相关性。使用其中野生型 GAPDH 被保留完整糖酵解活性的 H2O2 不敏感突变体取代的酵母菌株，我们证明 GAPDH 的 H2O2 敏感性是细胞对增加的 H2O2 水平的适应性反应的关键组成部分。
