A proton relay enhances H2O2 sensitivity of GAPDH to facilitate metabolic adaptation
A proton relay enhances H2O2 sensitivity of GAPDH to facilitate metabolic adaptation
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DOI：
10.1038/nchembio.1720
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发表时间：
2015-02-01
影响因子：
14.8
通讯作者：
Dick, Tobias P.
中科院分区：
文献类型：
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作者：
Peralta, David;Bronowska, Agnieszka K.;Dick, Tobias P.
3-磷酸甘油醛脱氢酶 (GAPDH) 对过氧化氢 (H2O2) 造成的可逆氧化失活敏感。在这里，我们展示了活性位点硫醇盐 (C152) 的 H2O2 反应性是由一种先前未认识到的机制催化的，该机制基于促进离去基团离开的专用质子中继。过氧化反应机制的破坏不会影响 GAPDH 的糖酵解活性。因此，特定且独立的机制分别介导相同硫醇盐亲核试剂对 H2O2 和甘油醛 3-磷酸的反应性。突变体的产生，其中 GAPDH 的糖酵解和过氧化活性完全解耦，可以直接评估 GAPDH H2O2 敏感性的生理相关性。使用将野生型 GAPDH 替换为保留完整糖酵解活性的 H2O2 不敏感突变体的酵母菌株，我们证明 GAPDH 的 H2O2 敏感性是细胞对 H2O2 水平增加的适应性反应的关键组成部分。
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.