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A proton relay enhances H2O2 sensitivity of GAPDH to facilitate metabolic adaptation

Nature Chemical Biology volume 11, pages 156–163 (2015)Cite this article

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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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Figure 1: C156 is dispensable for glycolysis, yet essential for efficient C152 oxidation.
Figure 2: Lack of C156 impairs S-glutathionylation of C152.
Figure 3: Computational chemistry suggests a mechanism for C152 oxidation by H2O2.
Figure 4: Site-directed mutagenesis supports the proton-shuttle mechanism.
Figure 5: GAPDH oxidation sensitivity contributes to cellular oxidative stress resistance.
Figure 6: A compensatory mutation restores oxidation sensitivity in the C156S mutant.

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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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Authors and Affiliations

  1. Division of Redox Regulation, DKFZ-ZMBH Alliance, German Cancer Research Center (DKFZ), Heidelberg, Germany

    David Peralta, Bruce Morgan, Koen Van Laer & Tobias P Dick

  2. Molecular Biomechanics, Heidelberg Institute of Theoretical Studies, Heidelberg, Germany

    Agnieszka K Bronowska & Frauke Gräter

  3. Faculty of Chemistry, University of Heidelberg, Heidelberg, Germany

    Agnieszka K Bronowska

  4. Department of Molecular Immunology and Toxicology, National Institute of Oncology, Budapest, Hungary

    Éva Dóka & Péter Nagy

  5. Department of Inorganic and Analytical Chemistry, University of Debrecen, Debrecen, Hungary

    Éva Dóka

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Contributions

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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Correspondence to Tobias P Dick.

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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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