Extended Data Fig. 1: Mutational inactivation of GAPDH oxidation sensitivity.

(a) Previously suggested proton relay mechanism facilitating GAPDH oxidation by H₂O₂. Cys-152 (C152) is the active site cysteine. The hydroxyl group of Tyr-314 (Y314) is required for the protonation of the hydroxide leaving group. The hydroxyl group of Thr-177 (T177) is thought to support the proper positioning of the Tyr-314 hydroxyl group. See Peralta et al., 2015, for further details. Dotted lines: hydrogen bonds. (b) Representative LC-MS/MS spectra of the GAPDH tryptic peptide containing the active site cysteine, before (lower panel) and after (upper panel) cellular H₂O₂ exposure, demonstrating selective S-glutathionylation of the active site cysteine under oxidative stress conditions. The calculated mass for fragment y11²⁺ of the glutathionylated peptide is 782.8 Da, the calculated mass of the corresponding alkylated fragment y11²⁺ is 701.8 Da. (c) Glutathionylation of GAPDH Cys-152 in WT and mutant cells, before and after treatment of intact cells with 50 µM H₂O₂ for 5 min, as determined by LC-MS/MS analysis. Same experiment as shown in main Fig. 1b, but including a second independently selected clone of the mutant cell line (Y314F_2). Bars represent the mean of 3 technical replicates. Representative of n = 3 biological replicates. (d) Hyperoxidation (sulfinylation and/or sulfonylation) of GAPDH Cys-152 as visualized by immunoblotting before and after treatment of intact cells with up to 2 mM H₂O₂ for 5 min. Same experiment as shown in main Fig. 1c, but including additional H₂O₂ concentrations and a second independently selected clone of the mutant cell line (Y314F_2). Representative of n = 3 biological replicates. (e) Outline of the strategy to determine relative flux of [1,2-¹³C]glucose into glycolysis and the pentose phosphate pathway (PPP), respectively. The ratio of singly and doubly labeled fructose-6-phosphate is taken to indicate relative PPP flux. (f) Extracellular acidification rate (ECAR) of cells expressing either WT or mutant GAPDH in response to 10 mM glucose (black arrow) and 100 µM H₂O₂ (red arrow). Based on n = 6 biological replicates. Dotted lines: SD.

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