Bell J E, Dalziel K
Biochim Biophys Acta. 1975 Jun 24;391(2):249-58. doi: 10.1016/0005-2744(75)90248-x.
The binding of NAD+, NADH and adenosine diphosphoribose (Ado-PP-Rib) to a stable, highly active and nucleotide-free preparation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) has been studied. All three nucleotides quench the protein fluorescence to the same extent when they bind to the enzyme, and this property has been used to measure the dissociation constants for the two high-affinity binding sites for the nucleotides. The results indicate negative interactions between, or non-identify of, these two binding sites, to which NAD+ and NADH bind with similar affinity. The binding of NAD+ to the enzyme has been studied by spectrophotometric titrations at 360 nm. It appears that the binding of NAD+ to each of the four subunits of the enzyme contributes equally to the intensity of this 'Racker' band. The dissociation constants associated with the binding of the third and fourth molecules of NAD+ estimated from such titrations confirm some previous estimates. The binding of NADH to the enzyme causes a decrease of intensity of the absorbance of the coenzyme at 340 nm, and the dissociation constants for binding of the third and fourth molecules of NADH have been estimated from spectrophotometric titrations. They are the same as those for NAD+. Judging by the apparent dissociation constants, negative interactions on binding the third molecule of NAD+ or NADH are more marked than those associated with the binding of the second and fourth molecules, suggesting that a major conformational change occurs at half-saturation of the tetramer with coenzyme.
已对烟酰胺腺嘌呤二核苷酸（NAD⁺）、还原型烟酰胺腺嘌呤二核苷酸（NADH）和腺苷二磷酸核糖（Ado-PP-Rib）与兔肌肉甘油醛-3-磷酸脱氢酶（D-甘油醛-3-磷酸：NAD⁺氧化还原酶（磷酸化），EC 1.2.1.12）的稳定、高活性且无核苷酸制剂的结合进行了研究。当这三种核苷酸与该酶结合时，它们对蛋白质荧光的淬灭程度相同，并且利用这一特性测定了核苷酸两个高亲和力结合位点的解离常数。结果表明这两个结合位点之间存在负相互作用或不相同，NAD⁺和NADH以相似的亲和力与它们结合。通过在360 nm处的分光光度滴定研究了NAD⁺与该酶的结合。看来NAD⁺与该酶的四个亚基中每个亚基的结合对这个“拉克尔”带的强度贡献相等。从这种滴定估计的与第三和第四个NAD⁺分子结合相关的解离常数证实了一些先前的估计。NADH与该酶的结合导致辅酶在340 nm处吸光度强度降低，并且通过分光光度滴定估计了第三和第四个NADH分子结合的解离常数。它们与NAD⁺的解离常数相同。从表观解离常数判断，结合第三个NAD⁺或NADH分子时的负相互作用比与第二个和第四个分子结合时的负相互作用更明显，这表明在四聚体被辅酶半饱和时发生了主要的构象变化。
