L-Ascorbic Acid: A True Substrate for HIF Prolyl Hydroxylase?
Cell Biology and Anatomy
L-Ascorbate (L-Asc), but not D-isoascorbate (D-Asc) and N-acetylcysteine (NAC) suppress HIF1 ODD-luc reporter activation induced by various inhibitors of HIF prolyl hydroxylase (PHD). The efficiency of suppression by L-Asc was sensitive to the nature of HIF PHD inhibitor chosen for reporter activation. In particular, the inhibitors developed to compete with alpha-ketoglutarate (alphaKG), were less sensitive to suppression by the physiological range of L-Asc (40-100muM) than those having a strong iron chelation motif. Challenging those HIF activators in the reporter system with D-Asc demonstrated that the D-isomer, despite exhibiting the same reducing potency with respect to ferric iron, had almost no effect compared to L-Asc. Similarly, no effect on reporter activation was observed with cell-permeable reducing agent NAC up to 1mM. Docking of L-Asc and D-Asc acid into the HIF PHD2 crystal structure showed interference of Tyr310 with respect to D-Asc. This suggests that L-Asc is not merely a reducing agent preventing enzyme inactivation. Rather, the overall results identify L-Asc as a co-substrate of HIF PHD that may compete for the binding site of alphaKG in the enzyme active center. This conclusion is in agreement with the results obtained recently in cell-based systems for TET enzymes and jumonji histone demethylases, where L-Asc has been proposed to act as a co-substrate and not as a reducing agent preventing enzyme inactivation.
Osipyants, A., Poloznikov, A., Smirnova, N., Hushpulian, D., Khristichenko, A., Chubar, T., Zakhariants, A., Ahuja, M., Gaisina, I., Thomas, B., Brown, A., Gazaryan, I., & Tishkov, V. (2018). L-Ascorbic Acid: A True Substrate for HIF Prolyl Hydroxylase?. Biochimie, 147, 46-54. https://doi.org/10.1016/j.biochi.2017.12.011