Purification and properties of NADP-linked glucose-6-phosphate dehydrogenase from Acetobacter hansenii (Acetobacter xylinum).
Purification and properties of NADP-linked glucose-6-phosphate dehydrogenase from Acetobacter hansenii (Acetobacter xylinum).
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汉森醋杆菌 (Acetobacter xylinum) 中 NADP 连接的葡萄糖-6-磷酸脱氢酶的纯化和特性。
DOI：
10.1016/0003-9861(91)90119-4
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发表时间：
1991
影响因子：
3.9
通讯作者：
Cook,C
中科院分区：
文献类型：
--
作者：
Levy,HR;Cook,C
从汉逊醋杆菌（Acetobacter hansenii）（以前称为木醋杆菌（Acetobacter xylinum））中纯化出NADP连接的葡萄糖-6-磷酸脱氢酶。测定了10个N-末端氨基酸的序列。十二烷基硫酸钠聚丙烯酰胺凝胶电泳测定该酶的亚基分子量为53，000;非变性条件下的凝胶过滤研究表明，在pH 6.5和9.5时，该酶的分子量为200，000至220，000，这表明天然酶是一种四聚体。在pH 6.5和9.5下的特异性研究表明，该酶是典型的NADP偏好葡萄糖-6-磷酸脱氢酶。该酶的催化活性随pH值的增加而增加，kcat在pH9.5时比pH6.7时约大4倍，而对NADP+的Km在较高pH值时低3倍;但葡萄糖6-磷酸的Km在pH9.5时比在pH6.7时高近20倍，表明该酶在较低pH时催化效率更高。在pH6.7时，初始速度测量，NADPH的产物抑制和葡糖胺6-磷酸的抑制产生与稳态随机机制一致的结果。在pH 9.5，稳态动力学分析表明，该机制是有序的，辅酶结合第一，但非线性双倒数图观察到NADPH的存在下，当葡萄糖6-磷酸变化，并没有进行完整的动力学分析。在所检测的几种核苷酸和潜在的抑制配体中，只有2′，5 ′-ADP对该酶有明显的抑制作用。
The NADP-linked glucose-6-phosphate dehydrogenase fromAcetobacter hansenii(formerly known asAcetobacter xylinum) has been purified to apparent homogeneity. The sequence of the 10 N-terminal amino acids was determined. The subunit molecular weight of the enzyme is 53,000 as determined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis; gel filtration studies under nondenaturing conditions revealed that the molecular weight of the enzyme is 200,000 to 220,000 at pH 6.5 and 9.5, suggesting that the native enzyme is a tetramer. Specificity studies at both pH 6.5 and 9.5 demonstrated that the enzyme is a typical NADP-preferring glucose-6-phosphate dehydrogenase. The enzyme's catalytic activity increases with increasing pH,kcatbeing approximately 4 times greater at pH 9.5 than at pH 6.7 and theKmfor NADP+being 3 times lower at the higher pH; but theKmfor glucose 6-phosphate is nearly 20 times higher at pH 9.5 than at pH 6.7, suggesting that the enzyme is catalytically more efficient at the lower pH. At pH 6.7, initial velocity measurements, product inhibition by NADPH, and inhibition by glucosamine 6-phosphate yielded results that were consistent with a steady-state random mechanism. At pH 9.5, steady-state kinetic analyses suggested that the mechanism is ordered, with coenzyme binding first, but nonlinear double-reciprocal plots were observed in the presence of NADPH when glucose 6-phosphate was varied and a complete kinetic analysis was not undertaken. Among several nucleotides and potential inhibitory ligands examined, only 2′,5′-ADP inhibited the enzyme significantly.