Copper Activates a Redox Switch to Reversibly Inhibit Glyceraldehyde-3-Phosphate Dehydrogenase

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), one of the most conserved proteins across all kingdoms of life, has a multitude of moonlighting functions beyond its enzymatic role in glycolysis. Metal binding to GAPDH has previously been reported to inhibit enzymatic activity in several prokaryotic and eukaryotic systems, although the mechanism of inhibition has not been elucidated. In this study, we examined the effects of zinc, silver, and copper ions on <i>Escherichia coli</i> GAPDH (<i>ec</i>GAPDH) and explore the mechanism of inhibition via enzymatic activity assays under aerobic and anaerobic conditions, electron paramagnetic spectroscopy, and mass spectrometry. This study shows that Zn²⁺ does not affect <i>ec</i>GAPDH activity, while Cu²⁺ causes redox inactivation that oxidizes the protein upon reduction to Cu⁺. Cu⁺ binds tightly to the protein (log <i>K</i>_a = 15.2 ± 0.2, pH 7.4), with diminished affinity in the presence of G3P substrate. Although the anaerobic binding of Cu⁺ or Ag⁺ moderately diminishes catalytic turnover, these ions sensitize the protein to rapid and complete oxidative inactivation in the presence of oxygen. Oxidative modification of the active site cysteine, including glutathionylation, is reversible. This oxidative process, which occurs upon exposure to Cu and Ag, bestows GAPDH the ability to act as an all-purpose redox switch responsive to toxic metals as well as reactive oxygen species. This work provides insight into shared mechanisms by which cells use redox inactivation of sentinel enzymes like GAPDH to redirect metabolic processes for cellular protection.