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 (H 2 O 2 ) 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 H 2 O 2 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 H 2 O 2 -induced inactivation of GAPDH. To explore this hypothesis, we systematically investigated GAPDH inactivation after exposure to physiologically-relevant H 2 O 2 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 H 2 O 2 -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.