Beth A H, Robinson B H, Cobb C E, Dalton L R, Trommer W E, Birktoft J J, Park J H
J Biol Chem. 1984 Aug 10;259(15):9717-28.
The spatial arrangement of coenzyme NAD+ in remote and adjacent binding sites in various stoichiometric complexes with tetrameric glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle was examined via EPR spectroscopy. An adenosine N6-15N,2H17 spin-labeled derivative of coenzyme NAD+ (SL-NAD+) was chemically synthesized for this work. The spectral simplifications and narrow line widths afforded by 15N and 2H substitution enabled experimental EPR spectra to be deconvoluted into their three component spectra: (a) unbound coenzyme, (b) bound coenzyme without adjacent site occupied, and (c) bound coenzyme with adjacent site occupied. Binding of SL-NAD+ in adjacent active centers of R axis-related subunits resulted in resolved dipolar interactions which characterized intersubunit distances. Binding to distant subunits related by the P and Q axes gave no dipolar interaction. Once the first NAD+ site was occupied, EPR spectra at various stoichiometries provided evidence for nonpreferential spatial binding of SL-NAD+ to the three unoccupied sites. EPR spectral simulations indicated a separation of 12.8 A for the unpaired electrons of spin label moieties of R axis-related coenzymes. Molecular modeling based on x-ray crystallographic data predicted 11-13 A. The angles and distance relating to interacting spin-labels were calculated from atomic coordinates based on molecular modeling of both anti-anti and anti-syn (adenine-ribose) conformations of SL-NAD+. Computer-generated line shapes indicated best agreement with experimental EPR results when the anti-anti geometry was employed. Comparison of EPR spectra from soluble and ammonium sulfate-precipitated enzymes indicated that the NAD+-binding domains are positioned equivalently in the two physical states. Since the observed dipolar line shapes are critically dependent on the distance and geometry relating to the interacting SL-NAD+, these data provide direct evidence for a high degree of conservation of quaternary structure of the enzyme in the hydrated crystalline state. Studies on the enzyme isolated from human erythrocytes also indicated a close correlation with the rabbit muscle enzyme in both the arrangement of NAD+-binding domains and negative cooperativity of coenzyme binding.
通过电子顺磁共振光谱法（EPR）研究了辅酶NAD⁺在与来自兔肌肉的四聚体甘油醛-3-磷酸脱氢酶形成的各种化学计量复合物中的远程和相邻结合位点的空间排列。为此工作化学合成了辅酶NAD⁺的腺苷N6-¹⁵N,²H¹⁷自旋标记衍生物（SL-NAD⁺）。¹⁵N和²H取代带来的光谱简化和窄线宽使实验EPR光谱能够解卷积为其三个组分光谱：（a）未结合的辅酶，（b）相邻位点未被占据的结合辅酶，以及（c）相邻位点被占据的结合辅酶。SL-NAD⁺在R轴相关亚基的相邻活性中心的结合导致了解析的偶极相互作用，其表征了亚基间距离。与P轴和Q轴相关的远距离亚基的结合未产生偶极相互作用。一旦第一个NAD⁺位点被占据，各种化学计量下的EPR光谱就为SL-NAD⁺与三个未占据位点的非优先空间结合提供了证据。EPR光谱模拟表明，R轴相关辅酶的自旋标记部分的未配对电子之间的间距为12.8 Å。基于X射线晶体学数据的分子建模预测为11 - 13 Å。根据SL-NAD⁺的反-反和顺-反（腺嘌呤-核糖）构象的分子建模，从原子坐标计算出与相互作用的自旋标记相关的角度和距离。当采用反-反几何结构时，计算机生成的线形表明与实验EPR结果最佳吻合。来自可溶性酶和硫酸铵沉淀酶的EPR光谱比较表明，NAD⁺结合结构域在两种物理状态下的定位相同。由于观察到的偶极线形严重依赖于与相互作用的SL-NAD⁺相关的距离和几何结构，这些数据为水合晶体状态下酶的四级结构高度保守提供了直接证据。对从人红细胞中分离出的酶的研究还表明，在NAD⁺结合结构域的排列和辅酶结合的负协同性方面，与人兔肌肉酶密切相关。
