In situ regeneration of NADH via lipoamide dehydrogenase-catalyzed electron transfer reaction evidenced by spectroelectrochemistry.
In situ regeneration of NADH via lipoamide dehydrogenase-catalyzed electron transfer reaction evidenced by spectroelectrochemistry.
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DOI：
10.1016/j.bioelechem.2012.03.002
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发表时间：
2012-08
影响因子：
5
通讯作者：
Liu, Jun
中科院分区：
文献类型：
--
作者：
Tam, Tsz Kin;Chen, Baowei;Lei, Chenghong;Liu, Jun
NAD/NADH是一种存在于所有活细胞中的辅酶，将电子从一个反应带到另一个反应。用紫外可见光谱电化学法研究了硫酰胺脱氢酶(LD)催化的电子转移反应原位再生NADH的特性。在1 mm厚的薄层光谱电化学池中，以金丝网为工作电极，在-0.75V(vs.Ag/AgCl.)电压下测得NADH再生的米氏常数(Km)和最大再生速度(Vmax)分别为0.80±0.15 mm和1.91±0.09μM S-1。进一步将NAD体系的电催化还原与乳酸脱氢酶催化丙酮酸转化为乳酸的反应相结合，考察了再生NADH的辅酶活性。虽然与NADH的电催化氧化相比，NAD的电催化还原为NADH是困难的，但我们的光谱电化学结果表明，通过LD催化的电子转移反应原位再生NADH是快速和可持续的，可以潜在地应用于许多NAD/NADH依赖的酶系统。
NAD/NADH is a coenzyme found in all living cells, carrying electrons from one reaction to another. We report on characterizations of in situ regeneration of NADH via lipoamide dehydrogenase (LD)-catalyzed electron transfer reaction to regenerate NADH using UV-vis spectroelectrochemistry. The Michaelis-Menten constant (Km) and maximum velocity (Vmax) of NADH regeneration were measured as 0.80 ± 0.15 mM and 1.91 ± 0.09 μM s-1 in a 1-mm thin-layer spectroelectrochemical cell using gold gauze as the working electrode at the applied potential -0.75 V (vs. Ag/AgCl). The electrocatalytic reduction of the NAD system was further coupled with the enzymatic conversion of pyruvate to lactate by lactate dehydrogenase to examine the coenzymatic activity of the regenerated NADH. Although the reproducible electrocatalytic reduction of NAD into NADH is known to be difficult compared to the electrocatalytic oxidation of NADH, our spectroelectrochemical results indicate that the in situ regeneration of NADH via LD-catalyzed electron transfer reaction is fast and sustainable and can be potentially applied to many NAD/NADH-dependent enzyme systems.
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