ACETOBACTER XYLINUM GLUCOSE 6-PHOSPHATE DEHYDROGENASES

醋酸杆菌木糖葡萄糖 6-磷酸脱氢酶

• 批准号: 3299155
• 负责人: H RICHARD LEVY
• 金额: $ 13.68万
• 依托单位: SYRACUSE UNIVERSITY AT SYRACUSE
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-12-01 至 1991-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3299155
• 关键词: biochemical evolution; chemical binding; chemical structure function; cofactor; conformation; enzyme mechanism; enzyme structure; glucose 6 phosphate dehydrogenase; structural genes

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），包括那些