Environmental factors modulate the responsiveness and reversibility of the H₂O₂-induced inactivation of GAPDH

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serves as a critical metabolic switch. GAPDH inactivation by hydrogen peroxide (H₂O₂) reroutes the carbon flux towards the pentose phosphate pathway to generate NADPH for antioxidant defence enzymes. However, a significant discrepancy exists between the modest in vitro sensitivity of GAPDH to H₂O₂ with its rapid inactivation within cells. We hypothesized that two commonly neglected characteristics of the intracellular milieu - macromolecular crowding and the presence of bicarbonate - would modulate H₂O₂-induced inactivation of GAPDH. To explore this hypothesis, we systematically investigated GAPDH inactivation after exposure to physiologically-relevant H₂O₂ doses, comparing dilute buffered conditions to systems containing bicarbonate and an inert high molecular mass polymer to simulate the molecular crowding present in vivo. Effects of the substrate and cofactor on enzyme inactivation were also quantified. Enzyme activity assays revealed that both crowded conditions and the presence of bicarbonate significantly enhanced H₂O₂-induced inactivation, with NAD⁺ further enhancing this responsiveness. LC-MS/MS data support the oxidation of the catalytic cysteine to an intramolecular disulfide bond which results in a loss of activity but prevents irreversible inactivation. The disulfide involving the catalytic cysteine showed an increase in the total occupancy from 3.2 % under dilute conditions to 11.6 % and 31.8 % under crowded + bicarbonate conditions in the absence and presence of 400 μM NAD⁺, respectively. This work bridges the gap in terms of responsiveness and reversibility of a key metabolic enzyme that serves as redox switch highlighting how physico-chemical (crowding) and chemical (bicarbonate, NAD⁺) environmental factors determine metabolic regulation under oxidative stress.