A proton relay enhances H2O2 sensitivity of GAPDH to facilitate metabolic adaptation

The metabolic enzyme GAPDH exhibits oxidative inactivation in response to H 2 O 2 . A proton relay system was identified that enhances H 2 O 2 sensitivity of GAPDH distinct from its catalytic activity, which ensures viability under oxidative stress. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is sensitive to reversible oxidative inactivation by hydrogen peroxide (H 2 O 2 ). Here we show that H 2 O 2 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 H 2 O 2 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 H 2 O 2 sensitivity. Using yeast strains in which wild-type GAPDH was replaced with H 2 O 2 -insensitive mutants retaining full glycolytic activity, we demonstrate that H 2 O 2 sensitivity of GAPDH is a key component of the cellular adaptive response to increased H 2 O 2 levels.