Structures of human cytosolic NADP-dependent isocitrate dehydrogenase reveal a novel self-regulatory mechanism of activity
Structures of human cytosolic NADP-dependent isocitrate dehydrogenase reveal a novel self-regulatory mechanism of activity
复制标题
人胞质 NADP 依赖性异柠檬酸脱氢酶的结构揭示了一种新的自我调节活性机制
DOI：
10.1074/jbc.m404298200
复制
发表时间：
2004-08-06
影响因子：
4.8
通讯作者：
Ding, JP
中科院分区：
文献类型：
--
作者：
Xu, X;Zhao, JY;Ding, JP
异柠檬酸脱氢酶（IDHs）催化异柠檬酸氧化脱羧生成α-酮戊二酸，IDHs酶活性的调节对其生物学功能至关重要。细菌的IDHs通过活性位点上一个严格保守的丝氨酸残基的磷酸化进行可逆调节。真核生物中依赖NADP的IDHs（NADP - IDHs）已被证明具有多种重要的生物学功能；然而，它们的调节机制仍不清楚。人胞质NADP - IDH（HcIDH）与NADP结合以及与NADP、异柠檬酸和Ca²⁺结合的结构研究揭示了该酶三种具有生物学相关性的构象状态，其活性位点的结构和整体结构存在显著差异。在所有已知的NADP - IDH结构中，活性位点处形成一个保守α-螺旋的结构片段在HcIDH的开放、无活性形式中呈现为环构象；在半开放的中间形式中为部分解开的α-螺旋；在关闭的活性形式中为α-螺旋。该片段的Asp(279)侧链占据异柠檬酸结合位点，在无活性形式中与Ser(94)（等同于细菌IDHs中的磷酸化位点）形成氢键，并在活性形式中螯合金属离子。结构数据使我们提出了一种HcIDH的新型自我调节机制，该机制模拟细菌同源物所使用的磷酸化机制，与生化和生物学数据相符。这种机制可能适用于其他真核生物的NADP - IDHs。这些结果还为真核生物NADP - IDHs对底物和辅因子的识别及特异性提供了见解。
Isocitrate dehydrogenases (IDHs) catalyze the oxidative decarboxylation of isocitrate to alpha-ketoglutarate, and regulation of the enzymatic activity of IDHs is crucial for their biological functions. Bacterial IDHs are reversibly regulated by phosphorylation of a strictly conserved serine residue at the active site. Eukaryotic NADP-dependent IDHs (NADP-IDHs) have been shown to have diverse important biological functions; however, their regulatory mechanism remains unclear. Structural studies of human cytosolic NADP-IDH (HcIDH) in complex with NADP and in complex with NADP, isocitrate, and Ca2+ reveal three biologically relevant conformational states of the enzyme that differ substantially in the structure of the active site and in the overall structure. A structural segment at the active site that forms a conserved alpha-helix in all known NADP-IDH structures assumes a loop conformation in the open, inactive form of HcIDH; a partially unraveled alpha-helix in the semi-open, intermediate form; and an alpha-helix in the closed, active form. The side chain of Asp(279) of this segment occupies the isocitrate-binding site and forms hydrogen bonds with Ser(94) ( the equivalent of the phosphorylation site in bacterial IDHs) in the inactive form and chelates the metal ion in the active form. The structural data led us to propose a novel self-regulatory mechanism for HcIDH that mimics the phosphorylation mechanism used by the bacterial homologs, consistent with biochemical and biological data. This mechanism might be applicable to other eukaryotic NADP-IDHs. The results also provide insights into the recognition and specificity of substrate and cofactor by eukaryotic NADP-IDHs.