Elevation of intracellular Zn2+ following ischemia contributes to cell death by affecting mitochondrial function. Zn2+ is a differential regulator of the mitochondrial enzyme lipoamide dehydrogenase (LADH) at physiological concentrations (Ka = 0.1 µM free zinc), inhibiting lipoamide and accelerating NADH dehydrogenase activities. These differential effects have been attributed to coordination of Zn2+ by LADH active-site cysteines. A detailed kinetic mechanism has now been developed for the diaphorase (NADH-dehydrogenase) reaction catalyzed by pig heart LADH using 2,6-dichlorophenol-indophenol (DCPIP) as a model quinone electron acceptor. Anaerobic stopped-flow experiments show that two-electron reduced LADH is 15-25-fold less active towards DCPIP reduction than four-electron reduced enzyme, or Zn2+-modified reduced LADH (the corresponding values of the rate constants are (6.5 ± 1.5) × 103 M–1·s–1, (9 ± 2) × 104 M–1·s–1, and (1.6 ± 0.5) × 105 M–1·s–1, respectively). Steady-state kinetic studies with different diaphorase substrates show that Zn2+ accelerates reaction rates exclusively for two-electron acceptors (duroquinone, DCPIP), but not for one-electron acceptors (benzoquinone, ubiquinone, ferricyanide). This implies that the two-electron reduced form of LADH, prevalent at low NADH levels, is a poor two-electron donor compared to the four-electron reduced or Zn2+-modified reduced LADH forms. These data suggest that zinc binding to the active-site thiols switches the enzyme from one- to two-electron donor mode. This zinc-activated switch has the potential to alter the ratio of superoxide and H2O2 generated by the LADH oxidase activity.

中文翻译：

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猪心硫辛酰胺脱氢酶中的锌开关：心肌黄酶反应的稳态和瞬态动力学研究

缺血后细胞内 Zn2+ 的升高通过影响线粒体功能导致细胞死亡。Zn2+ 在生理浓度（Ka = 0.1 µM 游离锌）下是线粒体酶硫辛酰胺脱氢酶 (LADH) 的差异调节剂，可抑制硫辛酰胺并加速 NADH 脱氢酶活性。这些不同的效应归因于 LADH 活性位点半胱氨酸对 Zn2+ 的配位。现已开发出一种详细的动力学机制，用于使用 2,6-二氯苯酚-吲哚酚 (DCPIP) 作为模型醌电子受体由猪心 LADH 催化的心肌黄酶（NADH-脱氢酶）反应。厌氧停流实验表明，两电子还原的 LADH 对 DCPIP 还原的活性比四电子还原酶低 15-25 倍，或 Zn2+ 修饰的还原 LADH（速率常数的相应值为 (6.5 ± 1.5) × 103 M–1·s–1、(9 ± 2) × 104 M–1·s–1 和 (1.6 ± 0.5 ) × 105 M–1·s–1，分别)。对不同心肌黄酶底物的稳态动力学研究表明，Zn2+ 仅能加快双电子受体（duroquinone、DCPIP）的反应速率，但不能加快单电子受体（苯醌、泛醌、铁氰化物）的反应速率。这意味着与四电子还原或 Zn2+ 修饰的还原 LADH 形式相比，在低 NADH 水平下普遍存在的两电子还原形式的 LADH 是较差的两电子供体。这些数据表明与活性位点硫醇结合的锌将酶从单电子供体模式转换为双电子供体模式。