Structure and Function of Quinoprotein Dehydrogenase

本文研究了大肠杆菌的吡咯喹啉醌依赖的奎诺蛋白脱氢酶、葡萄糖脱氢酶（GDH）以及恶臭假单胞菌和亚氧葡萄糖杆菌的两种醇脱氢酶（ADH）的结构和功能。1)大肠杆菌GDH的结构和功能研究：采用随机诱变和定点诱变制备了几种突变酶，并对其纯化后的反应动力学和氧化还原谱进行了检测。与结构模型比较，确定了His-262、His-775、Trp-404、Asp-466、Asp-730和Lys-493等氨基酸残基的功能。2)对恶臭p.p . putida ADH （ADH IIB）的电子转移研究：从恶臭p.p . putida的可溶部分中分离纯化ADH IIB与蓝铜蛋白azurin，并通过动力学、荧光和氧化还原滴定分析对两者之间的反应进行了研究。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和自由可逆的打开和关闭结合过程发生的。此外，纯化的细胞色素氧化酶也可以在膜泡上重建蓝蛋白依赖的醇氧化活性。3) E. coli GDH和G. subxydans ADH的泛素反应位点：通过与具有不同深度泛素类似物的磷脂重构GDH，发现GDH的泛素结合位点位于细胞质膜表面附近。在G. subxydans 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离。4) P. putida ADH和G. subboxydans ADH的X射线晶体学：P. putida ADH IIB的结构已经在1.9 A处成功测定。然而，G. subxydans ADH已经结晶，现在正在确定结构。

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.