Yamada M, Inbe H, Tanaka M, Sumi K, Matsushita K, Adachi O
Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan.
J Biol Chem. 1998 Aug 21;273(34):22021-7. doi: 10.1074/jbc.273.34.22021.
Several mutants of quinoprotein glucose dehydrogenase (GDH) in Escherichia coli were obtained and characterized. Of these, significant mutants were further characterized by kinetic analysis after purification or by site-directed mutagenesis to introduce different amino acid substitutions. H775R and H775A showed a pronounced reduction of affinity for a prosthetic group, pyrroloquinoline quinone (PQQ), suggesting that His-775 may directly interact with PQQ. D730N and D730A showed low glucose oxidase activity without influence on the affinity for PQQ, Mg2+, or substrate, but D730R showed reduced affinity for PQQ. The spectrum of tryptophan fluorescence revealed that the local structure surrounding PQQ was not changed by D730N mutation. Based on these data, we assume that Asp-730 may occur close to PQQ and function as a proton (and also electron) donor to PQQ or acceptor from PQQH2. Substitutions of Gly-689, that are located at the end of a unique segment of GDH among homologous quinoprotein dehydrogenases, directed reduction of the affinity for PQQ or GDH activity. Therefore, the unique segment and Asp-730 may play a specific role for GDH, which might be related to the intramolecular electron transfer from PQQ to ubiquinone.
我们获得并表征了大肠杆菌中喹啉蛋白葡萄糖脱氢酶（GDH）的几个突变体。其中，通过纯化后的动力学分析或通过定点诱变引入不同氨基酸取代对重要突变体进行了进一步表征。H775R和H775A对辅基吡咯并喹啉醌（PQQ）的亲和力显著降低，这表明His-775可能直接与PQQ相互作用。D730N和D730A表现出低葡萄糖氧化酶活性，且对PQQ、Mg2+或底物的亲和力不受影响，但D730R对PQQ的亲和力降低。色氨酸荧光光谱显示，D730N突变不会改变PQQ周围的局部结构。基于这些数据，我们推测Asp-730可能靠近PQQ，并作为质子（也是电子）供体给PQQ或从PQQH2接受质子。位于同源喹啉蛋白脱氢酶中GDH独特区段末端的Gly-689的取代导致对PQQ的亲和力降低或GDH活性降低。因此，独特区段和Asp-730可能对GDH发挥特定作用，这可能与从PQQ到泛醌的分子内电子转移有关。
