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ACETOBACTER XYLINUM GLUCOSE 6-PHOSPHATE DEHYDROGENASES

醋酸杆菌木糖葡萄糖 6-磷酸脱氢酶

基本信息

批准号：
3299156
负责人：
H RICHARD LEVY
金额：
$ 12.81万
依托单位：
SYRACUSE UNIVERSITY AT SYRACUSE
依托单位国家：
美国
项目类别：
财政年份：
1988
资助国家：
美国
起止时间：
1988-12-01 至 1992-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3299156
关键词：
biochemical evolution chemical binding chemical structure function cofactor conformation enzyme mechanism enzyme structure glucose 6 phosphate dehydrogenase structural genes

项目摘要

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.

大多数葡萄糖6-磷酸脱氢酶(G6PD)，包括在动物和高等植物的组织中发现，利用NADP作为他们的辅酶。一些细菌的G6PD，就像来自肠系膜明串珠菌可以利用NADP或NAD，各自不同的生理目的需要辅酶。这个木酸醋杆菌产生两个G6PD，一个专用于 NADP和其他需要NAD的。后者是唯一的NAD- 已知特定的G6PD。我们研究的长期目标是了解这些G6PD之间的结构差异与它们不同的辅酶特性有关机制化和规制化特征。《公约》的具体目标目前的建议是对比两个A的结构木聚糖酶G6PDs，特别是它们的辅酶结合部位，以及它们的动力和调节机制。两种酶都会纯净到同质。编码这两种酶的基因将被克隆到一株特殊构建的大肠杆菌中缺乏G6PD和DNA将被测序。此信息将用来推断两个G6PD是由发散还是趋同进化。稳态动力学机制，动力学和NAD和NADP的结合常数，以及调节特征这两种酶都会被测定。辅酶结合部位这两种酶将被共价标记和修饰含有该标签的多肽将被分离和测序。转移核Overhaser效应测量将用于评估与NAD-和NAD-结合的NAD+和NADP构象分别为11ADP连接的G6PD。这些研究与以下方面有关了解人类G6PD的机制和调控。人类G6PD之间存在相当大的序列同源性红细胞和肠系膜乳杆菌。尽管人类的G6PD是正常情况下，NADP特异，在一些遗传性疾病中，G6PD显示与NAD相关的高活性。某些类固醇是有效的抑制剂。人和动物的G6PD，而不是细菌的G6PD。可比性拟议的结构、机制和监管研究应会有所帮助阐明人类G6PDs异常的结构基础。