Morris C A, Weber M M
J Biol Chem. 1975 Apr 10;250(7):2681-9.
A soluble NAD+-linked isocitrate dehydrogenase has been isolated from Crithidia fasciculata. The enzyme was purified 128-fold, almost to homogeneity, and was highly specific for NAD+ as the coenzyme. There is also a cytoplasmic NADP+-linked and a mitochondrial isocitrate dehydrogenase in the organism. Studies of the physical and kinetic properties of the soluble NAD+-isocitrate dehydrogenase from this organism showed that it resembled microbial NADP+-isocitrate dehydrogenases in general, all of which are cytoplasmic enzymes. The enzyme appeared not to be related to other NAD+-isocitrate dehydrogenases, which are found in the mitochondria of eukaryotic cells. The molecular weight of the soluble NAD+-isocitrate dehydrogenase was 105,000 which is within the range of the values for microbial NADP+-isocitrate dehydrogenases. Similar to the NADP+-isocitrate dehydrogenase in this organism, the enzyme was inhibited in a concerted manner by glyoxalate plus oxalacetate. Kinetic analysis revealed that Mn2+ was involved in the binding of isocitrate to the enzyme. Inhibition of the NAD+-linked isocitrate dehydrogenase by p-chloromercuribenzoate could be prevented by prior incubation of the enzyme with both Mn2+ and isocitrate; however, neither ion alone conferred protection. Free isocitrate, free Mn2+, and the Mn2+-isocitrate complex could all bind to the enzyme. Four different mechanisms with respect to the binding of isocitrate to the enzyme were tested. Of these, the formation of the active enzyme-Mn2+-isocitrate complex from (a) the random binding of Mn2+, isocitrate, and the Mn2+-isocitrate complex, or (b) the binding of Mn2+-isocitrate with free Mn2+ and isocitrate acting as dead-end competitors were both in agreement with these data.
已从粪蝇锥虫中分离出一种可溶性的与NAD⁺相关的异柠檬酸脱氢酶。该酶被纯化了128倍，几乎达到了同质状态，并且对作为辅酶的NAD⁺具有高度特异性。该生物体中还存在一种细胞质中与NADP⁺相关的异柠檬酸脱氢酶以及一种线粒体异柠檬酸脱氢酶。对来自该生物体的可溶性NAD⁺-异柠檬酸脱氢酶的物理和动力学性质的研究表明，它总体上类似于微生物NADP⁺-异柠檬酸脱氢酶，所有这些都是细胞质酶。该酶似乎与真核细胞线粒体中发现的其他NAD⁺-异柠檬酸脱氢酶无关。可溶性NAD⁺-异柠檬酸脱氢酶的分子量为105,000，处于微生物NADP⁺-异柠檬酸脱氢酶的分子量范围内。与该生物体中的NADP⁺-异柠檬酸脱氢酶类似，该酶受到乙醛酸加草酰乙酸的协同抑制。动力学分析表明，Mn²⁺参与异柠檬酸与该酶的结合。对氯汞苯甲酸对NAD⁺相关异柠檬酸脱氢酶的抑制作用可通过先将该酶与Mn²⁺和异柠檬酸一起孵育来防止；然而，单独的任何一种离子都不能提供保护作用。游离的异柠檬酸、游离的Mn²⁺以及Mn²⁺-异柠檬酸复合物都可以与该酶结合。测试了关于异柠檬酸与该酶结合的四种不同机制。其中，由（a）Mn²⁺、异柠檬酸和Mn²⁺-异柠檬酸复合物的随机结合，或（b）Mn²⁺-异柠檬酸与游离Mn²⁺的结合以及异柠檬酸作为终产物竞争者的结合形成活性酶-Mn²⁺-异柠檬酸复合物，这两种情况都与这些数据一致。
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