OXIDATIVE MODIFICATION OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE

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)下降了50%。由于 这种酶在新陈代谢中的中心作用，重要的是要确定 导致这种酶活性丧失的因素。一件有希望的事 这可能是由于氧化修饰，一种过程 最近被认为与年龄相关的活动丧失有关 一些酵素。 我们发现肠系膜明串珠菌的G-6-PDH是 被微摩尔浓度的亚铁和铁迅速灭活 过氧化氢。失活与形成One相关 每个酶亚基的羰基官能度，表明失活是 这是特定部位氧化修饰的结果。我们的结果表明 亚铁结合到葡萄糖-6磷酸结合部位， 酶结合亚铁与过氧化氢引线的相互作用 对酶活性至关重要的氨基酸的氧化修饰。 部分失活的酶主要以二聚体形式存在， 其余活性亚基的表观亲和力没有变化 观察底物的变化。然而，部分失活确实导致了 剩余活性的热稳定性降低。这一下降 在很大程度上可以通过添加 葡萄糖-6-磷酸。因此，虽然接触过氧化氢和亚铁 铁导致G-6-PDH不可逆失活，从而 修饰是选择性的，导致两者的杂二聚体的形成 活性和非活性亚基，似乎不会引起大规模 结构性变化。 我们的结果证明了G-6-PDH的固有敏感性 从系膜乳杆菌到已知存在的氧化系统的修饰 在活体内。对铁的生理意义的评价 铁催化氧化G-6-PDH等待这些研究扩展到 已知的哺乳动物来源积累不太活跃或不活跃的形式的 酶是年龄的函数。

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.