OXIDATIVE MODIFICATION OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE

6-磷酸葡萄糖脱氢酶的氧化修饰

• 批准号: 3857971
• 负责人: L I SZWEDA
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3857971
• 关键词: active sites; cell age; chemical related neoplasm /cancer; dimer; enzyme mechanism; enzyme structure; enzyme substrate; eukaryote; glucose 6 phosphate dehydrogenase; hydrogen peroxide; oxidation

As a variety of eukaryotic cells age, the specific activity of glucose- 6-phosphate dehydrogenase (G-6-PDH) declines as much as 50%. Due to the central role of this enzyme in metabolism, it is important to define factors responsible for this loss in enzyme activity. One promising possibility is that this may be due to oxidative modification, a process which has recently been implicated in the age-related loss in activity of a number of enzymes. We have found that G-6-PDH from Leuconostoc mesenteroides is rapidly inactivated by micromolar concentrations of ferrous iron and hydrogen peroxide. Inactivation correlated with the formation of one carbonyl functionality per enzyme subunit, indicating that inactivation is the result of site-specific oxidative modification. Our results suggest that ferrous iron binds to the glucose-6phosphate binding site and that interaction of the enzyme-bound ferrous iron with hydrogen peroxide leads to the oxidative modification of amino acids essential for enzyme activity. Partially inactivated enzyme remained predominantly in the dimeric form and no change in the apparent affinity of the remaining active subunits for substrate was observed. Partial inactivation did, however, lead to a decrease in the thermal stability of the remaining activity. This decrease in thermal stability could be largely overcome by the addition of glucose-6-phosphate. Thus, while exposure to hydrogen peroxide and ferrous iron results in the irreversible inactivation of G-6-PDH, the resulting modification is selective, leads to the formation of heterodimers of both active and inactive subunits, and does not appear to cause large-scale structural changes. Our results demonstrate the inherent susceptibility of G-6-PDH from L. mesenteroides to modification by an oxidation system known to exist in vivo. An assessment of the physiological significance of ferrous iron-catalyzed oxidation of G-6-PDH awaits extension of these studies to mammalian sources known to accumulate less active or inactive forms of the enzyme as a function of age.

随着各种真核细胞的老化，葡萄糖的比活性- 6-磷酸脱氢酶（G-6-PDH）下降多达50%。 由于 这种酶在代谢中的中心作用，重要的是要确定 导致这种酶活性丧失的因素。 一个有希望 这可能是由于氧化修饰， 它最近被牵连在与年龄有关的活动的损失， 一些酶。 我们已经发现肠膜明串珠菌的G-6-PDH是 通过微摩尔浓度的亚铁快速灭活， 过氧化氢 失活与一个的形成相关 每个酶亚基的羰基官能度，表明失活是 位点特异性氧化修饰的结果。 我们的研究结果表明 亚铁与葡萄糖-6-磷酸结合位点结合， 酶结合亚铁与过氧化氢的相互作用 涉及酶活性所必需的氨基酸的氧化修饰。 部分失活的酶主要保持二聚体形式， 其余活性亚基对 观察底物。 然而，部分失活确实导致了 剩余活性的热稳定性降低。 这种减少 在热稳定性方面，可以通过添加 葡萄糖-6-磷酸 因此，当暴露于过氧化氢和亚铁 铁导致G-6-PDH的不可逆失活， 修饰是选择性的，导致两者的异二聚体的形成。 活性和非活性亚基，并且似乎不会引起大规模的 结构变化。 我们的研究结果表明，G-6-PDH的固有易感性 距离L.肠膜通过已知存在的氧化系统进行修饰 in vivo. 铁的生理意义的评价 G-6-PDH的铁催化氧化有待这些研究的扩展， 已知的哺乳动物来源积累活性较低或无活性形式的 酶与年龄的关系。