辅酶和底物与牛心线粒体中烟酰胺腺嘌呤二核苷酸磷酸连接的异柠檬酸脱氢酶的平衡结合

Equilibrium binding of coenzymes and substrates to nicotinamide-adenine dinucleotide phosphate-linked isocitrate dehydrogenase from bovine heart mitochondria.

Reynolds C H, Kuchel P W, Dalziel K

Biochem J. 1978 Jun 1;171(3):733-42. doi: 10.1042/bj1710733.

Abstract

The stoicheiometries and affinities of ligand binding to isocitrate dehydrogenase were studied at pH 7.0, mainly by measuring changes in NADPH and protein fluorescence. 2. The affinity of the enzyme for NADPH is about 100-fold greater than it is for NADP+ in various buffer/salt solutions, and the affinities for both coenzymes are decreased by Mg2+, phosphate and increase in ionic strength. 3. The maximum binding capacity of the dimeric enzyme for NADPH, from coenzyme fluorescence and protein-fluorescence measurements, and also for NADP+, by ultrafiltration, is 2 mol/mol of enzyme. Protein-fluorescence titrations of the enzyme with NADP+ are apparently inconsistent with this conclusion, indicating that the increase in protein fluorescence caused by NADP+ binding is not proportional to fractional saturation of the binding sites. 4. Changes in protein fluorescence caused by changes in ionic strength and by the binding of substrates, Mg2+ or NADP+ (but not NADPH) are relatively slow, suggesting conformation changes. 5. In the presence of Mg2+, the enzyme binds isocitrate very strongly, and 2-oxoglutarate rather weakly. 6. Evidence is presented for the formation of an abortive complex of enzyme-Mg2+-isocitrate-NADPH in which isocitrate and NADPH are bound much more weakly than in their complexes with enzyme and Mg2+ alone. 7. The results are discussed in relation to the interpretation of the kinetic properties of the enzyme and its behaviour in the mitochondrion.

摘要

主要通过测量NADPH和蛋白质荧光的变化，在pH 7.0条件下研究了配体与异柠檬酸脱氢酶结合的化学计量关系和亲和力。2. 在各种缓冲液/盐溶液中，该酶对NADPH的亲和力比对NADP⁺的亲和力大约高100倍，并且Mg²⁺、磷酸盐以及离子强度的增加会降低对这两种辅酶的亲和力。3. 通过辅酶荧光和蛋白质荧光测量得到的二聚体酶对NADPH的最大结合容量，以及通过超滤得到的对NADP⁺的最大结合容量均为2摩尔/摩尔酶。用NADP⁺对该酶进行蛋白质荧光滴定的结果显然与这一结论不一致，这表明NADP⁺结合引起的蛋白质荧光增加与结合位点的分数饱和度不成正比。4. 离子强度变化以及底物、Mg²⁺或NADP⁺（而非NADPH）结合引起的蛋白质荧光变化相对较慢，这表明存在构象变化。5. 在Mg²⁺存在的情况下，该酶与异柠檬酸结合非常紧密，而与2-氧代戊二酸结合较弱。6. 有证据表明形成了酶-Mg²⁺-异柠檬酸-NADPH的无效复合物，其中异柠檬酸和NADPH的结合比它们单独与酶和Mg²⁺形成的复合物中的结合要弱得多。7. 结合该酶的动力学性质及其在线粒体中的行为对结果进行了讨论。