OXIDATIVE MODIFICATION OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE

这项研究的目标一直是找出影响 细胞成分的特性随年龄的变化。 体内产生的自由基有可能损害细胞 组件。这一过程被牵连到与年龄相关的 细胞功能受损。体外研究旨在评估 关键代谢酶对已知氧化条件的敏感性 因此，在活体内的存在对于理解衰老至关重要。 金属催化产生的自由基已被证明会导致 各种酶的定点修饰和失活。我们有 先前报道了葡萄糖-6-磷酸脱氢酶(Glu-6-PDH) 从肠系膜明串珠菌中分离出来的细菌能被亚铁迅速灭活 过氧化氢以特定地点的方式。在这一点的延伸中 工作中，我们发现这种酶与亚铁和 柠檬酸会导致该酶的氧依赖失活。我们的数据 提示柠檬酸亚铁络合物与葡萄糖-6-结合。 磷酸盐结合部位，然后与过氧化氢反应 在溶液中形成，导致氨基酸的氧化修饰 对于酶的活性是必不可少的。 我们也知道，活性氧物种很容易与膜相互作用。 脂类，通常会导致不饱和醛的形成。这些 醛很可能扩散到它们所在的细胞介质中 可用于与大量双分子的简单反应。4- 羟基-2-壬烯醛(Hne)是膜的主要产物之一。 过氧化。我们发现Glu-6-PDH与HNE的孵育 一种假性一级酶活性丧失。我们的数据表明 HNE对Glu-6-PDH的失活是HNE与 参与结合的赖氨酸残基的表氨基 葡萄糖-6-磷酸。 我们的结果证实了L。 对已知存在于体内的氧化条件的肠系膜 可能是氧化应激导致的产物。因此，这些研究 为当前以活体为重点的研究提供有价值的信息 实验旨在直接评估这些过程在 与年龄相关的细胞功能损害。

The goal of this research has been to identify factors which influence changes in the properties of cellular components as a function of age. Free radicals generated in vivo have the potential to damage cellular components. This process has been implicated in the age-related impairment of cellular function. In vitro studies designed to assess the susceptibility of key metabolic enzymes to oxidative conditions known to exist in vivo are therefore critical to the understanding of aging. Metal-catalyzed production of free radicals has been shown to result in site-specific modification and inactivation of various enzymes. We have previously reported that glucose-6-phosphate dehydrogenase (Glu-6-PDH) from Leuconostoc mesenteroides is rapidly inactivated by ferrous iron and hydrogen peroxide in a site-specific manner. In an extension of this work, we have found that incubation of this enzyme with ferrous iron and citrate results in oxygen-dependent inactivation of the enzyme. Our data suggest that the ferrous iron-citrate complex binds to the glucose-6- phosphate binding site and then undergoes reaction with hydrogen peroxide formed in solution leading to the oxidative modification of amino acids essential for enzyme activity. Reactive oxygen species are also known to readily interact with membrane lipids, often resulting in the formation of unsaturated aldehydes. These aldehydes are likely to diffuse into cellular media where they are available for facile reaction with a vast array of bimolecules. 4- Hydroxy-2-nonenal (HNE) is one of the major products of membrane peroxidation. We have found that incubation of Glu-6-PDH with HNE leads to a pseudo-first-order loss of enzyme activity. Our data indicate that inactivation of Glu-6-PDH by HNE is the result of the reaction of HNE with the epsilon-amino group of a lysine residue involved in the binding of glucose-6-phosphate. Our results demonstrate the inherent susceptibility of Glu-6-PDH from L. mesenteroides to oxidative conditions known to exist in vivo and identify likely products resulting from oxidative stress. These studies therefore provide valuable information for current research which focuses on in vivo experiments designed to directly assess the role of these processes in the age-related impairment of cellular function.