Sancho J, Gómez-Moreno C
Departamento de Bioquímica y Biología Molecular y Cellular, Facultad de Ciencias, Universidad de Zaragoza, Spain.
Arch Biochem Biophys. 1991 Jul;288(1):231-8. doi: 10.1016/0003-9861(91)90189-p.
The interaction of ferredoxin-NADP+ reductase from the cyanobacterium Anabaena variabilis with its substrates, NADP+ and ferredoxin, has been studied by difference absorption spectroscopy. Several structural analogs of NADP+ have been shown to form complexes the stabilities of which are strongly dependent on the ionic strength of the medium. In most cases the binding energy of these complexes and their difference absorption spectra are similar to those reported for the spinach enzyme. However, NADP+ perturbs the absorption spectra of the Anabaena and spinach enzymes in a different way. This difference has been shown to be related to the binding of the nicotinamide ring of NADP+ to the enzymes. These results are interpreted as being due to a different nicotinamide binding site in the two reductases. The enthalpic and entropic components of the Gibbs energy of formation of the NADP+ complex have been estimated. An increase in entropy on NADP+ binding seems to be the main source of stability for the complex. A shift of approximately 40 mV in the redox potential of the couple NADP+/NADPH has been observed to occur upon binding of NADP+ to the oxidized enzyme. This allows us to calculate the binding energy between the reductase and NADPH. The ability of the reductase, ferredoxin, and NADP+ to form a ternary complex indicates that the protein carrier binds to the reductase through a different site than that of the pyridine nucleotide.
通过差示吸收光谱法研究了多变鱼腥蓝细菌中ferredoxin-NADP⁺还原酶与其底物NADP⁺和铁氧化还原蛋白的相互作用。已证明NADP⁺的几种结构类似物能形成复合物，其稳定性强烈依赖于介质的离子强度。在大多数情况下，这些复合物的结合能及其差示吸收光谱与菠菜酶报道的相似。然而，NADP⁺以不同方式扰动鱼腥蓝细菌和菠菜酶的吸收光谱。已表明这种差异与NADP⁺的烟酰胺环与酶的结合有关。这些结果被解释为由于两种还原酶中烟酰胺结合位点不同。已估算出NADP⁺复合物形成吉布斯自由能的焓和熵分量。NADP⁺结合时熵的增加似乎是复合物稳定性的主要来源。观察到NADP⁺与氧化型酶结合时，NADP⁺/NADPH电对的氧化还原电位发生了约40 mV的偏移。这使我们能够计算还原酶与NADPH之间的结合能。还原酶、铁氧化还原蛋白和NADP⁺形成三元复合物的能力表明，蛋白质载体与还原酶的结合位点不同于吡啶核苷酸的结合位点。
