Structure and Function of Quinoprotein Dehydrogenase

点击翻译按钮获取中文摘要

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.