Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229, USA.
Biochemistry. 2011 Sep 27;50(38):8241-50. doi: 10.1021/bi201088m. Epub 2011 Aug 30.
Yeast NAD(+)-specific isocitrate dehydrogenase is an allosterically regulated octameric enzyme composed of four heterodimers of a catalytic IDH2 subunit and a regulatory IDH1 subunit. Despite structural predictions that the enzyme would contain eight isocitrate binding sites, four NAD(+) binding sites, and four AMP binding sites, only half of the sites for each ligand can be measured in binding assays. On the basis of a potential interaction between side chains of Cys-150 residues in IDH2 subunits in each tetramer of the enzyme, ligand binding assays of wild-type (IDH1/IDH2) and IDH1/IDH2(C150S) octameric enzymes were conducted in the presence of dithiothreitol. These assays demonstrated the presence of eight isocitrate and four AMP binding sites for the wild-type enzyme in the presence of dithiothreitol and for the IDH1/IDH2(C150S) enzyme in the absence or presence of this reagent, suggesting that interactions between sulfhydryl side chains of IDH2 Cys-150 residues limit access to these sites. However, only two NAD(+) sites could be measured for either enzyme. A tetrameric form of IDH (an IDH1(G15D)/IDH2 mutant enzyme) demonstrated half-site binding for isocitrate (two sites) in the absence of dithiothreitol and full-site binding (four sites) in the presence of dithiothreitol. Only one NAD(+) site could be measured for the tetramer under both conditions. In the context of the structure of the enzyme, these results suggest that an observed asymmetry between heterotetramers in the holoenzyme contributes to interactions between IDH2 Cys-150 residues and to half-site binding of isocitrate, but that a form of negative cooperativity may limit access to apparently equivalent NAD(+) binding sites.
酵母 NAD(+)特异性异柠檬酸脱氢酶是一种别构调节的八聚体酶，由四个催化 IDH2 亚基和一个调节 IDH1 亚基组成的异二聚体组成。尽管结构预测表明该酶将包含八个异柠檬酸结合位点、四个 NAD(+)结合位点和四个 AMP 结合位点，但在结合测定中只能测量每个配体的一半位点。基于酶的每个四聚体中 IDH2 亚基的 Cys-150 残基侧链之间的潜在相互作用，在存在二硫苏糖醇的情况下，对野生型（IDH1/IDH2）和 IDH1/IDH2(C150S)八聚体酶进行了配体结合测定。这些测定表明，在存在二硫苏糖醇的情况下，野生型酶存在八个异柠檬酸和四个 AMP 结合位点，而在不存在或存在该试剂的情况下，IDH1/IDH2(C150S)酶存在这些位点，表明 IDH2 Cys-150 残基的巯基侧链之间的相互作用限制了这些位点的进入。然而，对于任一种酶，只能测量两个 NAD(+)位点。在不存在二硫苏糖醇的情况下，IDH 的四聚体形式（IDH1(G15D)/IDH2 突变酶）表现出异柠檬酸的半位点结合（两个位点），而在存在二硫苏糖醇的情况下，表现出全位点结合（四个位点）。在这两种情况下，对于四聚体只能测量一个 NAD(+)位点。在酶的结构背景下，这些结果表明，在全酶的异四聚体之间观察到的不对称性有助于 IDH2 Cys-150 残基之间的相互作用和异柠檬酸的半位点结合，但可能存在负协同作用限制了对明显等效的 NAD(+)结合位点的进入。
