Luisi P L, Baici A, Bonner F J, Aboderin A A
Biochemistry. 1975 Jan 28;14(2):362-8. doi: 10.1021/bi00673a024.
This work reports on the interaction of the fluorescent nicotinamide 1,N6-ethenoadenine dinucleotide (epsilonNAD+) with horse liver alcohol dehydrogenase, octopine dehydrogenase, and glyceraldehyde-3-phosphate dehydrogenase from different sources (yeast, lobster muscle, and rabbit muscle). The coenzyme fluorescence is enhanced by a factor of 10-13 in all systems investigated. It is shown that this enhancement cannot be due to changes in the polarity of the environment upon binding, and that it must be rather ascribed to structural properties of the bound coenzyme. Although dynamic factors could also be important for inducing changes in the quantum yield of epsilonNAD+ fluorescence, the close similarity of the fluorescence enhancement factor in all cases investigated indicates that the conformation of bound coenzyme is rather invariant in the different enzyme systems and overwhelmingly shifted toward an open form. Dissociation constants for epsilonNAD+-dehydrogenases complexes can be determined by monitoring the coenzyme fluorescence enhancement or the protein fluorescence quenching. In the case of yeast glyceraldehyde-3-phosphate dehydrogenase at pH 7.0 and t = 20 degrees the binding plots obtained by the two methods are coincident, and show no cooperativity. The affinity of epsilonNAD+ is generally lower than that of NAD+, although epsilonNAD+ maintains most of the binding characteristics of NAD+. For example, it forms a tight complex with horse liver alcohol dehydrogenase and pyrazole, and with octopine dehydrogenase saturated by L-arginine and pyruvate. One major difference in the binding behavior of NAD+ and epsilonNAD+ seems to be present in the muscle glyceraldehyde-3-phosphate dehydrogenase. In fact, no difference was found for epsilon NAD+ between the affinities of the third and fourth binding sites. The results and implications of this work are compared with those obtained recently by other authors.
本研究报道了荧光烟酰胺1,N6-乙烯腺嘌呤二核苷酸（εNAD+）与来自不同来源（酵母、龙虾肌肉和兔肌肉）的马肝醇脱氢酶、章鱼碱脱氢酶和甘油醛-3-磷酸脱氢酶之间的相互作用。在所研究的所有系统中，辅酶荧光增强了10至13倍。结果表明，这种增强并非由于结合后环境极性的变化，而必须归因于结合辅酶的结构特性。尽管动力学因素对于诱导εNAD+荧光量子产率的变化也可能很重要，但在所研究的所有情况下，荧光增强因子的高度相似性表明，结合辅酶的构象在不同的酶系统中相当不变，并且绝大多数向开放形式转变。εNAD+-脱氢酶复合物的解离常数可以通过监测辅酶荧光增强或蛋白质荧光猝灭来确定。在pH 7.0和t = 20℃条件下的酵母甘油醛-3-磷酸脱氢酶中，通过两种方法获得的结合曲线是一致的，并且没有协同性。尽管εNAD+保持了NAD+的大部分结合特性，但其对εNAD+的亲和力通常低于对NAD+的亲和力。例如，它与马肝醇脱氢酶和吡唑形成紧密复合物，与被L-精氨酸和丙酮酸饱和的章鱼碱脱氢酶也形成紧密复合物。NAD+和εNAD+结合行为的一个主要差异似乎存在于肌肉甘油醛-3-磷酸脱氢酶中。事实上，对于εNAD+，第三和第四结合位点的亲和力没有差异。本文的结果和意义与其他作者最近获得的结果进行了比较。
