Peng Yingjie, Zhong Chen, Huang Wei, Ding Jianping
State Key Laboratory of Molecular Biology and Research Center for Structural Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
Protein Sci. 2008 Sep;17(9):1542-54. doi: 10.1110/ps.035675.108. Epub 2008 Jun 13.
Isocitrate dehydrogenases (IDHs) catalyze oxidative decarboxylation of isocitrate (ICT) into alpha-ketoglutarate (AKG). We report here the crystal structures of Saccharomyces cerevesiae mitochondrial NADP-IDH Idp1p in binary complexes with coenzyme NADP, or substrate ICT, or product AKG, and in a quaternary complex with NADPH, AKG, and Ca(2+), which represent different enzymatic states during the catalytic reaction. Analyses of these structures identify key residues involved in the binding of these ligands. Comparisons among these structures and with the previously reported structures of other NADP-IDHs reveal that eukaryotic NADP-IDHs undergo substantial conformational changes during the catalytic reaction. Binding or release of the ligands can cause significant conformational changes of the structural elements composing the active site, leading to rotation of the large domain relative to the small and clasp domains along two hinge regions (residues 118-124 and residues 284-287) while maintaining the integrity of its secondary structural elements, and thus, formation of at least three distinct overall conformations. Specifically, the enzyme adopts an open conformation when bound to NADP, a quasi-closed conformation when bound to ICT or AKG, and a fully closed conformation when bound to NADP, ICT, and Ca(2+) in the pseudo-Michaelis complex or with NADPH, AKG, and Ca(2+) in the product state. The conformational changes of eukaryotic NADP-IDHs are quite different from those of Escherichia coli NADP-IDH, for which significant conformational changes are observed only between two forms of the apo enzyme, suggesting that the catalytic mechanism of eukaryotic NADP-IDHs is more complex than that of EcIDH, and involves more fine-tuned conformational changes.
异柠檬酸脱氢酶（IDHs）催化异柠檬酸（ICT）氧化脱羧生成α-酮戊二酸（AKG）。我们在此报告酿酒酵母线粒体NADP-IDH Idp1p与辅酶NADP、底物ICT或产物AKG形成的二元复合物的晶体结构，以及与NADPH、AKG和Ca(2+)形成的四元复合物的晶体结构，这些结构代表了催化反应过程中的不同酶状态。对这些结构的分析确定了参与这些配体结合的关键残基。将这些结构与先前报道的其他NADP-IDHs的结构进行比较，发现真核NADP-IDHs在催化反应过程中经历了显著的构象变化。配体的结合或释放可导致构成活性位点的结构元件发生显著的构象变化，导致大结构域相对于小结构域和扣合结构域沿着两个铰链区（残基118 - 124和残基284 - 287）旋转，同时保持其二级结构元件的完整性，从而形成至少三种不同的整体构象。具体而言，该酶与NADP结合时呈开放构象，与ICT或AKG结合时呈准封闭构象，在假米氏复合物中与NADP、ICT和Ca(2+)结合或在产物状态下与NADPH、AKG和Ca(2+)结合时呈完全封闭构象。真核NADP-IDHs的构象变化与大肠杆菌NADP-IDH有很大不同，后者仅在两种无辅基酶形式之间观察到显著的构象变化，这表明真核NADP-IDHs的催化机制比EcIDH更复杂，且涉及更精细的构象变化。
