Isocitrate dehydrogenase 1 (IDH1) catalyzes the reversible NADP+-dependent conversion of isocitrate to α-ketoglutarate (αKG) to provide critical cytosolic substrates and drive NADPH-dependent reactions like lipid biosynthesis and glutathione regeneration. In biochemical studies, the forward reaction is studied at neutral pH, while the reverse reaction is typically characterized in more acidic buffers. This led us to question whether IDH1 catalysis is pH-regulated, which would have functional implications under conditions that alter cellular pH, like apoptosis, hypoxia, cancer, and neurodegenerative diseases. Here, we show evidence of catalytic regulation of IDH1 by pH, identifying a trend of increasing kcat values for αKG production upon increasing pH in the buffers we tested. To understand the molecular determinants of IDH1 pH sensitivity, we used the pHinder algorithm to identify buried ionizable residues predicted to have shifted pKa values. Such residues can serve as pH sensors, with changes in protonation states leading to conformational changes that regulate catalysis. We identified an acidic residue buried at the IDH1 dimer interface, D273, with a predicted pKa value upshifted into the physiological range. D273 point mutations had decreased catalytic efficiency and, importantly, loss of pH-regulated catalysis. Based on these findings, we conclude that IDH1 activity is regulated, at least in part, by pH. We show this regulation is mediated by at least one buried acidic residue ∼12 Å from the IDH1 active site. By establishing mechanisms of regulation of this well-conserved enzyme, we highlight catalytic features that may be susceptible to pH changes caused by cell stress and disease.

中文翻译：

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埋在异柠檬酸脱氢酶1（IDH1）二聚体界面中的酸性残基有助于调节催化作用和pH敏感性。

异柠檬酸脱氢酶1（IDH1）催化异柠檬酸向α-酮戊二酸（αKG）的可逆NADP +依赖性转化，以提供关键的胞质底物并驱动NADPH依赖性反应，如脂质生物合成和谷胱甘肽再生。在生化研究中，正反应是在中性pH下进行的，而逆反应通常是在更酸性的缓冲液中表征的。这使我们怀疑IDH1催化是否受pH调节，这在改变细胞pH的条件下会产生功能性影响，例如细胞凋亡，缺氧，癌症和神经退行性疾病。在这里，我们显示了通过pH催化IDH1催化调节的证据，确定了在我们测试的缓冲液中pH升高后，αKG产量的kcat值升高的趋势。要了解IDH1 pH敏感性的分子决定因素，我们使用pHinder算法来识别预测的pKa值已漂移的掩埋可电离残基。此类残留物可充当pH传感器，质子化状态的变化会导致调节催化的构象变化。我们确定了埋在IDH1二聚体界面D273上的酸性残基，其预测的pKa值上移至生理范围。D273点突变降低了催化效率，重要的是，失去了pH调节的催化作用。基于这些发现，我们得出结论，IDH1活性至少部分受pH调节。我们显示该调节是由来自IDH1活性位点的至少一个约12Å的酸性掩埋残基介导的。通过建立调节这种保守酶的机制，