Shinar E, Navok T, Chevion M
J Biol Chem. 1983 Dec 25;258(24):14778-83.
The mechanism of enzymatic inactivation of purified and membrane-bound acetylcholine esterase by ascorbate and copper was investigated. While the exposure of the enzyme to either ascorbate or copper did not cause enzymatic inactivation, the incubation of the enzyme with a combination of both ascorbate and copper resulted in a loss in acetylcholine esterase activity, which was time dependent. The enzymatic inactivation required either molecular oxygen or hydrogen peroxide under anaerobic conditions. Scavengers of hydroxyl radicals at concentrations of up to 100 mM did not provide protection to acetylcholine esterase. Only mannitol at very high concentrations (above 1 M) efficiently prevented the inactivation of the enzyme. The kinetics of the aerobic oxidation of reduced ascorbate in the presence of acetylcholine esterase and copper closely followed the rate of enzyme inactivation. Addition of the chelating agents EDTA and diethylenetriaminepentaacetic acid prevented both the oxidation of ascorbate and the inactivation of the enzyme. In the presence of low concentrations of histidine (0.5-2.0 mM), which forms high affinity complexes with copper, the rate of ascorbate oxidation was similar to that recorded in its absence. On the other hand, no enzyme inactivation was indicated in the presence of histidine. Low temperature EPR measurements have demonstrated the binding of copper to the enzyme, and have shown the reduction of the cupric enzyme to the corresponding cuprous complex. In view of these results, a general "site-specific" mechanism for biological damage can be offered, in which copper(II) ions are bound to enzymes or other biological macromolecules. Ascorbate plays a dual role: it reduces the cupric complex to the corresponding cuprous state and serves as a source for H2O2, which, in turn, reacts with the reduced copper complex, in a Fenton reaction. In this reaction, secondary hydroxyl radicals are site specifically formed, and react preferentially with the protein, at the site of their formation, causing its inactivation. This mechanism is analogous to that previously proposed (Samuni, A., Chevion, M., and Czapski, G. (1981) J. Biol. Chem. 256, 12632-12635) for the enhancement of the biological damage caused by superoxide in the presence of copper.
研究了抗坏血酸盐和铜对纯化的及膜结合的乙酰胆碱酯酶的酶促失活机制。虽然将酶单独暴露于抗坏血酸盐或铜中不会导致酶失活，但将酶与抗坏血酸盐和铜一起孵育会导致乙酰胆碱酯酶活性丧失，且这种失活具有时间依赖性。在厌氧条件下，酶促失活需要分子氧或过氧化氢。浓度高达100 mM的羟基自由基清除剂不能保护乙酰胆碱酯酶。只有非常高浓度（高于1 M）的甘露醇能有效防止酶失活。在乙酰胆碱酯酶和铜存在的情况下，还原型抗坏血酸盐的有氧氧化动力学与酶失活速率密切相关。添加螯合剂乙二胺四乙酸（EDTA）和二乙烯三胺五乙酸可防止抗坏血酸盐氧化和酶失活。在低浓度组氨酸（0.5 - 2.0 mM）存在的情况下，组氨酸会与铜形成高亲和力复合物，此时抗坏血酸盐的氧化速率与不存在组氨酸时记录的速率相似。另一方面，在组氨酸存在的情况下未显示酶失活。低温电子顺磁共振测量表明铜与酶结合，并显示出铜酶还原为相应的亚铜复合物。鉴于这些结果，可以提出一种普遍的“位点特异性”生物损伤机制，其中铜（II）离子与酶或其他生物大分子结合。抗坏血酸盐起着双重作用：它将铜复合物还原为相应的亚铜状态，并作为过氧化氢的来源，而过氧化氢又会在芬顿反应中与还原的铜复合物反应。在这个反应中，二级羟基自由基在特定位点形成，并在其形成位点优先与蛋白质反应，导致其失活。这种机制类似于之前提出的（萨穆尼，A.，切维翁，M.，和查普斯基，G.（1981年）《生物化学杂志》256，12632 - 12635）在铜存在下超氧化物增强生物损伤的机制。
