Bloemen Rebecca H J, Radi Rafael, Davies Michael J, Fuentes-Lemus Eduardo
Department of Biomedical Sciences, Panum Institute, University of Copenhagen, Blegdamsvej 3, Copenhagen 2200, Denmark.
Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo 11800, Uruguay.
Biochem J. 2024 Dec 4;481(23):1855-1866. doi: 10.1042/BCJ20240597.
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.
甘油醛-3-磷酸脱氢酶（GAPDH）中的活性位点半胱氨酸残基对过氧化氢（H₂O₂）氧化敏感，这会导致酶失活。这会使碳通量从糖酵解重新导向磷酸戊糖途径，有利于生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H₂O₂适度增加，且存在高浓度的替代（非信号）硫醇和高效的过氧化物清除系统时，关于GAPDH如何有效氧化和失活存在重大知识空白。因此，我们探究了GAPDH失活是否会因两个具有体内相关性的因素而增强：大分子拥挤，这是生物环境的固有特性；以及碳酸氢盐的存在，这是一种丰富的生物缓冲剂。已知碳酸氢盐可通过形成过氧一碳酸酯来调节H₂O₂代谢。在有无25 mM碳酸氢钠的情况下，在稀释和拥挤条件下（由惰性高分子聚合物和小分子诱导）用低剂量H₂O₂进行实验，评估GAPDH活性。观察到在大分子拥挤条件下，H₂O₂诱导的GAPDH失活显著增强，碳酸氢盐具有额外作用。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）的氧化还原开关机制中至关重要。
