Purification and characterization of the NAD-preferring glucose 6-phosphate dehydrogenase from Acetobacter hansenii (Acetobacter xylinum).
Purification and characterization of the NAD-preferring glucose 6-phosphate dehydrogenase from Acetobacter hansenii (Acetobacter xylinum).
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汉森醋杆菌 (Acetobacter xylinum) 中 NAD 偏好的葡萄糖 6-磷酸脱氢酶的纯化和表征。
DOI：
10.1006/abbi.1994.1179
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发表时间：
1994
影响因子：
3.9
通讯作者：
Levy,HR
中科院分区：
文献类型：
--
作者：
Ragunathan,S;Levy,HR
汉逊醋杆菌（Acetobacter hansenii，以前称为木醋杆菌（Acetobacter xylinum））的NAD偏好性葡萄糖6-磷酸脱氢酶已被纯化至表观均一性并进行了动力学表征。通过使用甘油、MgSO 4和2-巯基乙醇在pH 5.4下稳定纯化的酶。用非变性凝胶过滤法测得酶的分子量为243，000。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测定的亚基分子量为60，140，表明天然酶是四聚体。在pH5.4时，该酶对NAD+和NADP+的Km值分别为0.104和0.34 mM;对葡萄糖6-磷酸的Km值分别为0.071和0.089 mM;对NAD+和NADP+的kcat值分别为128，000和77，300 min− 1。NADP+和葡萄糖6-磷酸的Km值比同一生物体中NADP特异性葡萄糖6-磷酸脱氢酶的Km值高约10倍，但kcat也高约10倍，因此在pH5.4时这两种活性的kcat/Km值几乎相同。NAD-偏好酶的NAD-和NADP-连接的活性都被ATP抑制。NADP特异性葡萄糖6-磷酸脱氢酶在pH 6.7和9.5时对ATP不敏感，但在pH 5.4时ATP抑制该酶。这两种葡萄糖6-磷酸脱氢酶在A.汉斯尼尔讨论道。
An NAD-preferring glucose 6-phosphate dehydrogenase ofAcetobacter hansenii(formerly known asAcetobacter xylinum) has been purified to apparent homogeneity and kinetically characterized. The purified enzyme was stabilized by the use of glycerol, MgSO4, and 2-mercaptoethanol at pH 5.4. The molecular weight of the enzyme, determined by nondenaturing gel filtration, is 243,000. The subunit molecular weight is 60,140 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the native enzyme is a tetramer. At pH 5.4 the enzyme hasKms of 0.104 and 0.34 mM for NAD+and NADP+, respectively; theKms for glucose 6-phosphate are 0.071 and 0.089 mM, using NAD+and NADP+, respectively; and thekcatvalues are 128,000 and 77,300 min−1with NAD+and NADP+, respectively. TheKms for NADP+and glucose 6-phosphate are approximately 10 times higher than the correspondingKms for the NADP-specific glucose 6-phosphate dehydrogenase in the same organism, but thekcatis also approximately 10-fold higher, so that thekcat/Kmvalues for these two activities are nearly identical at pH 5.4. Both the NAD- and NADP-linked activities of the NAD-preferring enzyme are inhibited by ATP. The NADP-specific glucose 6-phosphate dehydrogenase is insensitive to ATP at pH 6.7 and 9.5, but at pH 5.4 ATP inhibits this enzyme. The possible roles of these two glucose 6-phosphate dehydrogenases in the metabolism ofA. hanseniiare discussed.