Activity of select dehydrogenases with sepharose-immobilized N(6)-carboxymethyl-NAD.
Activity of select dehydrogenases with sepharose-immobilized N(6)-carboxymethyl-NAD.
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DOI：
10.1080/21655979.2014.1004020
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发表时间：
2015
期刊：
影响因子：
4.9
通讯作者：
Vieille C
中科院分区：
文献类型：
--
作者：
Beauchamp J;Vieille C
N6-羧甲基-NAD（N6-CM-NAD）可用于将NAD结合到含有末端伯胺的底物上。我们先前将N6-CM-NAD固定到琼脂糖珠上，并表明海栖热袍菌甘油脱氢酶可以使用固定化辅因子进行辅因子循环。我们现在表明，酿酒酵母醇脱氢酶，兔肌肉L-乳酸脱氢酶（XI型），牛肝L-谷氨酸脱氢酶（III型），肠膜明串珠菌葡萄糖-6-磷酸脱氢酶，和海栖热袍菌甘露醇脱氢酶与可溶性N6-CM-NAD的活性。琼脂糖固定化的N6-CM-NAD经T. Maritima甘油脱氢酶，表明N6-固定化的NAD适用于各种不同的脱氢酶。酶的活性位点的观察表明，空间位阻发挥更大的作用，在限制或允许活性与修饰的辅因子比极性和电荷的残基周围的N6-胺基NAD。
N6-carboxymethyl-NAD (N6-CM-NAD) can be used to immobilize NAD onto a substrate containing terminal primary amines. We previously immobilized N6-CM-NAD onto sepharose beads and showed that Thermotoga maritima glycerol dehydrogenase could use the immobilized cofactor with cofactor recycling. We now show that Saccharomyces cerevisiae alcohol dehydrogenase, rabbit muscle L-lactate dehydrogenase (type XI), bovine liver L-glutamic dehydrogenase (type III), Leuconostoc mesenteroides glucose-6-phosphate dehydro-genase, and Thermotoga maritima mannitol dehydrogenase are active with soluble N6-CM-NAD. The products of all enzymes but 6-phospho-D-glucono-1,5-lactone were formed when sepharose-immobilized N6-CM-NAD was recycled by T. maritima glycerol dehydrogenase, indicating that N6-immobilized NAD is suitable for use by a variety of different dehydrogenases. Observations of the enzyme active sites suggest that steric hindrance plays a greater role in limiting or allowing activity with the modified cofactor than do polarity and charge of the residues surrounding the N6-amine group on NAD.