Department of Cell Biology and Anatomy, New York Medical College, Valhalla, NY 10605, USA.
Department of Chemistry and Physical Sciences, Dyson College of Arts and Sciences, Pace University, Pleasantville, NY 10570, USA.
Biochemistry (Mosc). 2020 Aug;85(8):908-919. doi: 10.1134/S0006297920080064.
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 (K = 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) × 10 M·s, (9 ± 2) × 10 M·s, and (1.6 ± 0.5) × 10 M·s, 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.
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+ 是生理浓度下（游离锌的 K = 0.1 µM）线粒体酶硫辛酰胺脱氢酶 (LADH) 的差异调节剂，抑制硫辛酰胺并加速 NADH 脱氢酶的活性。这些差异效应归因于 LADH 活性部位半胱氨酸与 Zn2+ 的配位。现在已经为猪心 LADH 催化的二氢吩嗪（NADH-脱氢酶）反应开发了详细的动力学机制，使用 2,6-二氯苯酚靛酚（DCPIP）作为模型醌电子受体。厌氧停流实验表明，二电子还原的 LADH 对 DCPIP 还原的活性比四电子还原的酶或 Zn2+-修饰的还原的 LADH 低 15-25 倍（相应的速率常数值分别为 (6.5 ± 1.5) × 10 M·s、(9 ± 2) × 10 M·s 和 (1.6 ± 0.5) × 10 M·s）。用不同的二氢吩嗪底物进行稳态动力学研究表明，Zn2+ 仅加速二电子受体（二氢醌、DCPIP）的反应速率，而不加速一电子受体（苯醌、泛醌、铁氰化物）的反应速率。这意味着在低 NADH 水平下占优势的 LADH 的二电子还原形式与四电子还原或 Zn2+-修饰的还原 LADH 形式相比，是一种较差的二电子供体。这些数据表明，锌与活性部位巯基的结合将酶从单电子供体模式切换为双电子供体模式。这种锌激活的开关有可能改变 LADH 氧化酶活性产生的超氧化物和 H2O2 的比例。
