OXIDATIVE MODIFICATION OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE

本研究的目的是确定影响 作为年龄的函数的细胞成分的性质的变化。 体内产生的自由基有可能损害细胞 件. 这一过程与年龄有关， 细胞功能受损。 体外研究旨在评估 关键代谢酶对氧化条件的敏感性， 因此对理解衰老至关重要。 金属催化的自由基的产生已被证明会导致 位点特异性修饰和各种酶的失活。 我们有 先前报道，葡萄糖-6-磷酸脱氢酶（Glu-6-PDH） 来自肠膜明串珠菌的蛋白质被亚铁迅速灭活， 过氧化氢以特定的方式。 在这一延伸中， 工作，我们已经发现，这种酶与亚铁和 柠檬酸盐导致酶的氧依赖性失活。 我们的数据 表明亚铁-柠檬酸盐复合物与葡萄糖-6- 磷酸盐结合位点，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 对酶活性至关重要。 还已知活性氧物质容易与膜相互作用 脂质，通常导致不饱和醛的形成。 这些 醛可能扩散到它们所处的细胞介质中， 可用于与大量双分子的容易反应。 4- 羟基壬烯醛（hydroxy-2-nonenal，HNE）是膜的主要产品之一 过氧化作用 我们发现Glu-6-PDH与HNE的孵育 酶活性的伪一级损失。 我们的数据表明 HNE使Glu-6-PDH失活是HNE与 赖氨酸残基的ε-氨基，参与结合 葡萄糖-6-磷酸 我们的研究结果表明L. 肠膜的氧化条件已知存在于体内，并确定 可能是氧化应激的产物。 因此，这些研究 为目前的研究提供了有价值的信息， 实验旨在直接评估这些过程在 与年龄相关的细胞功能损伤。

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.