Anderson B M, Anderson C D
Department of Biochemistry and Anaerobic Microbiology, Virginia Polytechnic Institute and State University, Blacksburg 24061-0308, USA.
Arch Biochem Biophys. 1995 Aug 1;321(1):94-100. doi: 10.1006/abbi.1995.1372.
Azotobacter vinelandii glucose-6-phosphate dehydrogenase isolated from cell sonicates was purified 81-fold to electrophoretic homogeneity and a specific activity of 73 units/mg protein using ion-exchange and Matrex Dye chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and molecular exclusion chromatography indicated the enzyme to be a tetramer composed of 52,000 M(r) subunits. The enzyme utilized both NAD and NADP as coenzymes with Km values of 220 and 50 microM, respectively. In addition, the purified enzyme functioned well with the thionicotinamide analogs of NAD and NADP. A sigmoidal response was observed in studies of the effect of glucose 6-phosphate concentration on initial velocities. Evidence in support of one enzyme with dual coenzyme specificity was obtained in purification, thermodenaturation, and inhibitor studies. The enzyme exhibited a pH optimum of 8.5. Coenzyme-competitive inhibition was observed with nine adenosine derivatives with no significant selectivity shown for 2'-phosphoryl derivatives. Ki values for product inhibition by NADH and NADPH were higher than the Km values for the respective oxidized forms of the coenzymes.
从细胞超声破碎物中分离得到的维涅兰德固氮菌葡萄糖-6-磷酸脱氢酶，通过离子交换和Matrex染料层析法纯化了81倍，达到电泳纯，比活性为73单位/毫克蛋白。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和分子排阻层析表明该酶是由52,000 M(r)亚基组成的四聚体。该酶以NAD和NADP作为辅酶，Km值分别为220和50 microM。此外，纯化后的酶对NAD和NADP的硫代烟酰胺类似物也有良好的作用。在研究葡萄糖6-磷酸浓度对初始速度的影响时观察到了S形反应。在纯化、热变性和抑制剂研究中获得了支持一种具有双重辅酶特异性的酶的证据。该酶的最适pH为8.5。观察到九种腺苷衍生物对辅酶具有竞争性抑制作用，对2'-磷酸化衍生物没有明显的选择性。NADH和NADPH对产物抑制的Ki值高于相应辅酶氧化形式的Km值。
