Department of Chemistry and Biochemistry, San Diego State University, San Diego, CA 92182, U.S.A.
Harper Cancer Research Institute, Department of Chemistry and Biochemistry, University of Notre Dame, South Bend, IN 46617, U.S.A.
Biochem J. 2020 Aug 28;477(16):2999-3018. doi: 10.1042/BCJ20200311.
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.
异柠檬酸脱氢酶 1（IDH1）催化 NADP+-依赖性的异柠檬酸可逆转化为α-酮戊二酸（αKG），为细胞提供关键的细胞质底物，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，正向反应在中性 pH 下进行研究，而反向反应通常在更酸性的缓冲液中进行特征描述。这使我们质疑 IDH1 催化是否受 pH 调节，在改变细胞 pH 的条件下（如细胞凋亡、缺氧、癌症和神经退行性疾病），这将具有功能意义。在这里，我们证明了 pH 对 IDH1 催化的调节作用，在我们测试的缓冲液中，随着 pH 的增加，αKG 产生的 kcat 值呈增加趋势，这表明存在这种调节作用。为了了解 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 值发生变化的埋藏可离子化残基。这些残基可以作为 pH 传感器，质子化状态的变化导致构象变化，从而调节催化作用。我们鉴定出一个位于 IDH1 二聚体界面的酸性残基 D273，其预测的 pKa 值上移到生理范围内。D273 点突变降低了催化效率，更重要的是，丧失了 pH 调节的催化作用。基于这些发现，我们得出结论，IDH1 活性至少部分受到 pH 的调节。我们表明这种调节至少部分是由 IDH1 活性位点约 12 Å 处的一个埋藏酸性残基介导的。通过建立这种高度保守酶的调节机制，我们突出了可能对细胞应激和疾病引起的 pH 变化敏感的催化特征。
