ACETOBACTER XYLINUM GLUCOSE 6-PHOSPHATE DEHYDROGENASES

大多数葡萄糖6-磷酸脱氢酶（G6 PD），包括那些 在动物和高等植物的组织中发现，利用NADP作为 他们的辅酶。 一些细菌G6 PD，比如来自 肠膜明串珠菌可以利用NADP或NAD， 辅酶是不同生理目的所必需的。 的 木醋杆菌产生两种G6 PD，一种特异于 NADP和另一个需要NAD。 后者是唯一的NAD- 已知特定的G6 PD 我们研究的长期目标是 了解这些G6 PD之间的结构差异， 有助于它们不同的辅酶特异性， 机械和调节特征。 的具体目标 目前的建议是对比两个A的结构 木质素G6 PD，特别是其辅酶结合位点，和 它们的动力学和调节机制。 这两种酶都是 纯化至均一。 编码这两种酶的基因将 被克隆到一个特别构建的大肠杆菌菌株中 缺乏G6 PD的DNA将被测序。 这些信息将 可以用来推断这两次G6 PD是由分歧引起的， 趋同进化 稳态动力学机制，动力学 NAD和NADP的结合常数，以及调节特征 将对两种酶进行测定。 辅酶结合位点 将被共价标记，并且修饰的 含有标记的肽将被分离和测序。 转移核Overhauser效应测量将用于 评估NAD+和NADP与NAD-结合的构象， 11例ADP连锁的G6 PD。 这些研究与 了解人类G6 PD的机制和调节。 人的G6 PD与人的G6 PD之间存在相当大的序列同源性。 红细胞和L.肠系膜 虽然人类的G6 PD 通常是NADP特异性的，在某些遗传性疾病中，G6 PD显示 高NAD相关活性。 某些类固醇是有效的抑制剂 人类和动物的G6 PD，但不是细菌的G6 PD。 比较 建议进行的结构、机制及规管研究，应有助 阐明异常人类G6 PD的结构基础。

Most glucose 6-phosphate dehydrogenases (G6PDs), including those found in the tissues of animals and higher plants, utilize NADP as their coenzyme. A few bacterial G6PDs, like the one from Leuconostoc mesenteroides can utilize either NADP or NAD, each coenzyme being required for different physiological purposes. The bacterium Acetobacter xylinum produces two G6PDs, one specific for NADP and the other requiring NAD. The latter is the only NAD- specific G6PD known. The long-term goals of our research are to understand the structural differences among these G6PDs that contribute to their different coenzyme specificities and mechanistic and regulatory features. The specific aims of the present proposal are to contrast the structures of the two A xylinum G6PDs, especially of their coenzyme binding sites, and their kinetic and regulatory mechanisms. Both enzymes will be purified to homogeneity. The genes encoding both enzymes will be cloned into a specially constructed Escherichia coli strain lacking G6PD and the DNAs will be sequenced. This information will be used to infer whether the two G6PDs arose by divergent or convergent evolution. The steady-state kinetic mechanisms, kinetic and binding constants for NAD and NADP , and regulatory features will be determined for both enzymes. The coenzyme binding sites of both enzymes will be covalently labeled and the modified peptides containing the label will be isolated and sequenced. Transfer nuclear Overhauser effect measurements will be used to assess the conformations of NAD+ and NADP bound to the NAD- and 11ADP-linked G6PDs, respectively. These studies are relevant to understanding the mechanism and regulation of human G6PD. Considerable sequence homology exists between the G6PDs of human erythrocytes and L. mesenteroides. Although the human G6PD is normally NADP-specific, in some hereditary disorders G6PD shows high NAD-linked activity. Certain steroids are potent inhibitors of human and animal G6PD, but not bacterial G6PDs. The comparative structural, mechanistic and regulatory studies proposed should help to illuminate the structural basis of abnormal human G6PDs.