Abstract
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is sensitive to reversible oxidative inactivation by hydrogen peroxide (H2O2). Here we show that H2O2 reactivity of the active site thiolate (C152) is catalyzed by a previously unrecognized mechanism based on a dedicated proton relay promoting leaving group departure. Disruption of the peroxidatic reaction mechanism does not affect the glycolytic activity of GAPDH. Therefore, specific and separate mechanisms mediate the reactivity of the same thiolate nucleophile toward H2O2 and glyceraldehyde 3-phosphate, respectively. The generation of mutants in which the glycolytic and peroxidatic activities of GAPDH are comprehensively uncoupled allowed for a direct assessment of the physiological relevance of GAPDH H2O2 sensitivity. Using yeast strains in which wild-type GAPDH was replaced with H2O2-insensitive mutants retaining full glycolytic activity, we demonstrate that H2O2 sensitivity of GAPDH is a key component of the cellular adaptive response to increased H2O2 levels.
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Acknowledgements
T.P.D. is supported by the German Research Foundation (SFB 1036, SPP 1710). F.G. is supported by the Klaus Tschira Foundation. D.P. was supported by a PhD scholarship from the Helmholtz International Graduate School for Cancer Research. A.K.B. was supported by the Heidelberg University BIOMS program. P.N. is grateful for financial support from FP7-PEOPLE-2010-RG (Marie Curie International Reintegration Grant; grant no. PIRG08-GA-2010-277006), the Hungarian National Science Foundation (OTKA; grant no.: K 109843) and the EurocanPlatform project. T. Ruppert (DKFZ-ZMBH Alliance) is acknowledged for performing MS analysis. K. Ballagó and G. Kuntz are acknowledged for their technical assistance.
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D.P. and T.P.D. conceived the project. D.P. performed and analyzed most experiments. A.K.B. and F.G. designed and performed computational chemistry calculations. B.M. designed yeast experiments. E.D., K.V.L. and P.N. designed and performed kinetic experiments. All authors analyzed the data, discussed the results and contributed to the writing of the manuscript.
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Peralta, D., Bronowska, A., Morgan, B. et al. A proton relay enhances H2O2 sensitivity of GAPDH to facilitate metabolic adaptation. Nat Chem Biol 11, 156–163 (2015). https://doi.org/10.1038/nchembio.1720
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DOI: https://doi.org/10.1038/nchembio.1720
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