Structure and Function of Quinoprotein Dehydrogenase

本研究采用多种方法对大肠杆菌的葡萄糖脱氢酶（GDH）、恶臭假单胞菌（Pseudomonasputida）和弱氧化葡萄糖酸杆菌（Gluconacetylsuboxylans）的两种乙醇脱氢酶（ADH）进行了结构和功能的研究。1)对E. coli GDH：采用随机诱变和定点诱变的方法制备了几种突变酶，并对其纯化后的反应动力学和氧化还原光谱进行了研究。通过与结构模型的比较，确定了His-262、His-775、Trp-404、Asp-466、Asp-730和Lys-493氨基酸残基的功能。2)恶臭假单胞菌的电子传递研究ADH：从恶臭假单胞菌的可溶性组分中纯化ADH IIB和蓝铜蛋白天青蛋白，然后通过动力学、荧光和氧化还原滴定分析来检测两种蛋白之间的反应。结果表明，两种蛋白质之间的电子转移是通过疏水相互作用以及自由可逆的开和关结合过程发生的。此外，天青蛋白依赖的醇氧化活性可以重建膜囊泡，也与纯化的细胞色素氧化酶。3)E. coli GDH和G. subboxerans ADH：通过用具有不同深度的泛醌类似物的磷脂重构GDH，显示GDH的泛醌结合位点位于细胞质膜表面附近。In G. suboxanans ADH中，除了泛醌还原位点外，还发现泛醇氧化位点与泛醌还原位点分开存在。4）恶臭假单胞菌ADH和G. suboxanans ADH：恶臭假单胞菌ADH Ⅱ B的结构已在1.9 A处成功确定。而G. suboxanans ADH已结晶，目前正在测定结构。

Pyrroloquinoline quinone-dependent quinoprotein dehydrogenases, glucose dehydrogenase (GDH) of Escherichia coli and two alcohol dehydrogenases (ADH) of Pseudomonas putida and Gluconobacter suboxydans have been investigated to elucidate their structure and function, which was performed by several different means. 1)Structural and functional study of E. coli GDH : Several mutant enzymes were prepared by using random and site-directed mutageneses, and their reaction kinetics and oxido-reduction spectra were examined after purification. Compared with the structural model, the function of following amino acid residues, His-262, His-775, Trp-404, Asp-466, Asp-730 and Lys-493, have been identifisd. 2)Electron transfer study of P. putida ADH : (ADH IIB) was purified together with a blue copper protein, azurin, from the soluble fraction of P. putida and then the reaction between both proteins was examined by kinetic, fluorometric and redox titration analyses. As a result, the electron transfer between both proteins was shown to occur by a hydrophobic interaction and also by a freely reversible on and off binding process. Furthermore, the azurin-dependent alcohol oxidation activity could be reconstituted on the membrane vesicles and also with purified cytochrome oxidase. 3)Ubiquinone reaction site of E. coli GDH and G. suboxydans ADH : The ubiquinone-binding site of GDH was shown to be located near the surface of cytoplasmic membrane by reconstituting GDH with phospholipids having ubiquinone analogs at different depth. In G. suboxydans ADH, besides the ubiquinone reduction site, ubiquinol oxidation site was found to present separated from the site of ubiquinone reduction. 4)X ray crystallography of P. putida ADH and G. suboxydans ADH : The structure of ADH IIB of P. putida has already been successfully determined at 1.9 A. Whereas, G. suboxydans ADH has been crystallized and now under determination of the structure.