Zinc is a potent inhibitor of thiol oxidoreductase activity and stimulates reactive oxygen species production by lipoamide dehydrogenase
Zinc is a potent inhibitor of thiol oxidoreductase activity and stimulates reactive oxygen species production by lipoamide dehydrogenase
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DOI：
10.1074/jbc.m108264200
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发表时间：
2002-03-22
影响因子：
4.8
通讯作者：
Brown, AM
中科院分区：
文献类型：
--
作者：
Gazaryan, IG;Krasnikov, BF;Brown, AM
亚微摩尔锌抑制α-酮戊二酸依赖的线粒体呼吸。这归因于In。α-酮戊二酸脱氢酶复合体的抑制(Brown，A.M.，Kristal，B.S.，Effron，M.S.，Shestopalov，A.I.，UlLucci，P.A.，Sheu，K.-F.R.，Blass，J.P.，和Cooper，A.J.L.(2000)J.Biol.化学275,13441-13447)。脂酰胺脱氢酶是α-酮戊二酸脱氢酶复合体和另外两个线粒体复合体的一个组成部分，它催化还原等价物从相邻的二氢硫胺酰基转移酶亚基结合的二氢磷脂转移到NAD(+)。这个可逆反应涉及两个反应中心：一个是接受来自二氢硫酸盐的电子的硫醇对，另一个是将电子转移到NAD(+)的非共价结合的FAD部分。纯化的猪心酶催化的硫胺脱氢酶反应被锌离子(K-I类似于0.15微米)双向强烈抑制。稳态动力学研究表明，锌离子与氧化的硫胺竞争双电子还原酶。在厌氧停流实验中，直接检测到锌离子与双电子还原酶的相互作用。脂酰胺脱氢酶也催化NADH被氧气氧化，主要产物是过氧化氢，次要产物是超氧阴离子自由基。锌离子可使酶反应速度提高5倍，活化常数为0.09+/-0.02微米。活化是锌离子与还原的催化硫醇结合的结果，这阻止了催化二硫化物和FAD之间的还原当量的离域。建立了一个令人满意地描述观察到的影响的动力学方案，并将其应用于确定酶反应中的一些动力学参数。锌离子对不同LADH活性的不同影响代表了一种受金属离子结合调节的酶特异性可逆开关的新例子。这些结果表明，锌离子可以干扰线粒体抗氧化剂的产生，并可能通过一种新的机制刺激活性氧的产生。
Submicromolar zinc inhibits alpha-ketoglutarate-dependent mitochondrial respiration. This was attributed to in. hibition of the a-ketoglutarate dehydrogenase complex (Brown, A. M., Kristal, B.S., Effron, M.S., Shestopalov, A. I., Ullucci, P. A., Sheu, K.-F. R., Blass, J. P., and Cooper, A. J. L. (2000) J. Biol. Chem 275,13441-13447). Lipoamide dehydrogenase, a component of the alpha-ketoglutarate dehydrogenase complex and two other mitochondrial complexes, catalyzes the transfer of reducing equivalents from the bound dihydrolipoate of the neighboring dihydrolipoamide acyltransferase subunit to NAD(+). This reversible reaction involves two reaction centers: a thiol pair, which accepts electrons from dihydrolipoate, and a non-covalently bound FAD moiety, which transfers electrons to NAD(+). The lipoamide dehydrogenase reaction catalyzed by the purified pig heart enzyme is strongly inhibited by Zn2+ (K-i similar to0.15 muM) in both directions. Steady-state kinetic studies revealed that Zn2+ competes with oxidized lipoamide for the two-electron-reduced enzyme. Interaction of Zn2+ with the two-electron-reduced enzyme was directly detected in anaerobic stopped-flow experiments. Lipoamide dehydrogenase also catalyzes NADH oxidation by oxygen, yielding hydrogen peroxide as the major product and superoxide radical as a minor product. Zn2+ accelerates the oxidase reaction up to 5-fold with an activation constant of 0.09 +/- 0.02 muM. Activation is a consequence of Zn2+ binding to the reduced catalytic thiols, which prevents delocalization of the reducing equivalents between catalytic disulfide and FAD. A kinetic scheme that satisfactorily describes the observed effects has been developed and applied to determine a number of enzyme kinetic parameters in the oxidase reaction. The distinct effects of Zn2+ on different LADH activities represent a novel example of a reversible switch in enzyme specificity that is modulated by metal ion binding. These results suggest that Zn2+ can interfere with mitochondrial antioxidant production and may also stimulate production of reactive oxygen species by a novel mechanism.