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 (αKG), were less sensitive to suppression by the physiological range of L-Asc (40–100 μM) 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 1 mM. 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 αKG 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.

L-抗坏血酸盐（L-Asc），但不抑制D-异抗坏血酸盐（D-Asc）和N-乙酰半胱氨酸（NAC）抑制由各种HIF脯氨酰羟化酶（PHD）抑制剂诱导的HIF1 ODD-luc报告基因激活。L-Asc抑制的效率对选择用于报道分子激活的HIF PHD抑制剂的性质敏感。特别是，与α-酮戊二酸（αKG）竞争的抑制剂，对L-Asc（40-100μM）生理范围的抑制作用不如具有强铁螯合基序的抑制剂敏感。用D-Asc对报告系统中的HIF活化剂进行挑战表明，尽管D-异构体对铁的还原力相同，但与L-Asc相比几乎没有作用。类似地，使用细胞渗透性还原剂NAC（至多1 mM）也未观察到对报告基因激活的影响。L-Asc和D-Asc酸对接到HIF PHD2晶体结构中显示出Tyr310对D-Asc的干扰。这表明L-Asc不仅是防止酶失活的还原剂。相反，总体结果表明，L-Asc是HIF PHD的共底物，可竞争酶活性中心中αKG的结合位点。该结论与最近在基于细胞的TET酶和jumonji组蛋白脱甲基酶的系统中获得的结果一致，其中L-Asc被提议作为共底物而不是作为防止酶失活的还原剂。总体结果表明，L-Asc是HIF PHD的共底物，可以竞争酶活性中心中αKG的结合位点。该结论与最近在基于细胞的TET酶和jumonji组蛋白脱甲基酶的系统中获得的结果一致，其中L-Asc被提议作为共底物而不是作为防止酶失活的还原剂。总体结果表明，L-Asc是HIF PHD的共底物，可以竞争酶活性中心中αKG的结合位点。该结论与最近在基于细胞的TET酶和jumonji组蛋白脱甲基酶的系统中获得的结果一致，其中L-Asc被提议作为共底物而不是作为防止酶失活的还原剂。