Incubation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with micromolar hydrogen peroxide concentrations does not alter the catalytic properties of GAPDH in the reaction of oxidative phosphorylation of glyceraldehyde-3-phosphate, but endows the enzyme with the ability to catalyze the reaction in the absence of inorganic phosphate, producing NADH and 3-phosphoglycerate. The reaction is supposed to occur as a result of intramolecular acyl transfer from Cys-149 to a sulfenic acid form of Cys-153, followed by hydrolysis of the intermediate. The 'mildly oxidized' form of the enzyme can be easily converted back to the form unable to catalyze glyceraldehyde-3-phosphate oxidation in the absence of phosphate, by the addition of thiols.