The active site Cys residue in glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is sensitive to oxidation by hydrogen peroxide (H2O2), with this resulting in enzyme inactivation. This re-routes the carbon flux from glycolysis to the pentose phosphate pathway favoring the formation of NADPH and synthetic intermediates required for antioxidant defense and repair systems. Consequently, GAPDH inactivation serves as a redox switch for metabolic adaptation under conditions of oxidative stress. However, there is a major knowledge gap as to how GAPDH is efficiently oxidized and inactivated, when the increase in intracellular H2O2 is modest, and there is a high concentration of alternative (non-signaling) thiols and efficient peroxide removing systems. We have therefore explored whether GAPDH inactivation is enhanced by two factors of in vivo relevance: macromolecular crowding, an inherent property of biological environments, and the presence of bicarbonate, an abundant biological buffer. Bicarbonate is already known to modulate H2O2 metabolism via formation of peroxymonocarbonate. GAPDH activity was assessed in experiments with low doses of H2O2 under both dilute and crowded conditions (induced by inert high molecular mass polymers and small molecules), in both the absence and presence of 25 mM sodium bicarbonate. H2O2-induced inactivation of GAPDH was observed to be significantly enhanced under macromolecular crowding conditions, with bicarbonate having an additional effect. These data strongly suggest that these two factors are of major importance in redox switch mechanisms involving GAPDH (and possibly other thiol-dependent systems) within the cellular environment.

中文翻译：

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大分子拥挤和碳酸氢盐增强过氧化氢诱导的甘油醛-3-磷酸脱氢酶的失活。


甘油醛-3-磷酸脱氢酶 （GAPDH） 中的活性位点 Cys 残基对过氧化氢 （H2O2） 敏感，从而导致酶失活。这将碳通量从糖酵解重新路由到磷酸戊糖途径，有利于形成抗氧化防御和修复系统所需的 NADPH 和合成中间体。因此，GAPDH 失活在氧化应激条件下作为代谢适应的氧化还原开关。然而，当细胞内 H 2 O 2 的增加适度，并且存在高浓度的替代（非信号）硫醇和有效的过氧化物去除系统时，关于 GAPDH 如何被有效氧化和灭活存在重大知识空白。因此，我们探讨了 GAPDH 失活是否通过体内相关的两个因素得到增强：大分子拥挤，生物环境的固有特性，以及碳酸氢盐的存在，一种丰富的生物缓冲剂。众所周知，碳酸氢盐通过形成过氧单碳酸酯来调节 H 2 O 2 代谢。在稀和拥挤条件下（由惰性高分子量聚合物和小分子诱导）下，在不存在和存在 25 mM 碳酸氢钠的情况下，用低剂量 H 2 O 2 的实验评估了 GAPDH 活性。观察到 H 2 O 2 诱导的 GAPDH 失活在大分子拥挤条件下显着增强，碳酸氢盐具有额外的作用。这些数据强烈表明，这两个因素在细胞环境中涉及 GAPDH （可能还有其他硫醇依赖性系统） 的氧化还原转换机制中非常重要。