Pacific Northwest National Laboratory, Richland, WA 99352, USA.
Bioelectrochemistry. 2012 Aug;86:92-6. doi: 10.1016/j.bioelechem.2012.03.002. Epub 2012 Mar 27.
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 (K(m)) and maximum velocity (V(max)) 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.
NAD/NADH 是一种存在于所有活细胞中的辅酶，它将电子从一个反应传递到另一个反应。我们报告了通过脂酰脱氢酶 (LD) 催化的电子转移反应原位再生 NADH 的特性，使用紫外可见光谱电化学法再生 NADH。在 1 毫米厚的薄层光谱电化学池中，使用金网作为工作电极，在施加的 -0.75 V（相对于 Ag/AgCl）电势下，测量 NADH 再生的米氏常数 (K(m)) 和最大速度 (V(max)) 分别为 0.80±0.15 mM 和 1.91±0.09 μM s(-1)。NAD 系统的电催化还原进一步与乳酸脱氢酶催化的丙酮酸转化为乳酸偶联，以检查再生 NADH 的辅酶活性。尽管与 NADH 的电催化氧化相比，NAD 的可重复电催化还原已知是困难的，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，并可能应用于许多 NAD/NADH 依赖性酶系统。
