Szweda L I, Stadtman E R
Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1992 Feb 15;267(5):3096-100.
As a variety of eukaryotic cells age, the specific activity of glucose-6-phosphate dehydrogenase (Glu-6-PDH) declines as much as 50%. Because of the central role of this enzyme in metabolism, it is important to define factors responsible for this loss in enzyme activity. We report that Glu-6-PDH from Leuconostoc mesenteroides is rapidly inactivated by micromolar concentrations of Fe2+ and H2O2. Inactivation correlated with the formation of one carbonyl functionality/enzyme subunit, indicating that inactivation is the result of site-specific oxidative modification. Our results suggest that Fe2+ binds to the glucose 6-phosphate binding site and that interaction of the enzyme-bound Fe2+ with H2O2 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, although exposure to H2O2 and Fe2+ results in the irreversible inactivation of Glu-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 Glu-6-PDH from L. mesenteroides to modification by an oxidation system known to exist in vivo. An assessment of the physiological significance of Fe(2+)-catalyzed oxidation of Glu-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.
随着各种真核细胞老化，葡萄糖-6-磷酸脱氢酶（Glu-6-PDH）的比活性下降多达50%。由于该酶在代谢中的核心作用，确定导致这种酶活性丧失的因素很重要。我们报告，来自肠系膜明串珠菌的Glu-6-PDH会被微摩尔浓度的Fe2+和H2O2迅速灭活。灭活与每酶亚基一个羰基官能团的形成相关，表明灭活是位点特异性氧化修饰的结果。我们的结果表明，Fe2+与葡萄糖6-磷酸结合位点结合，并且酶结合的Fe2+与H2O2的相互作用导致对酶活性至关重要的氨基酸的氧化修饰。部分失活的酶主要仍以二聚体形式存在，并且未观察到剩余活性亚基对底物的表观亲和力发生变化。然而，部分失活确实导致剩余活性的热稳定性降低。通过添加葡萄糖6-磷酸，这种热稳定性的降低在很大程度上可以被克服。因此，尽管暴露于H2O2和Fe2+会导致Glu-6-PDH不可逆地失活，但所产生的修饰是选择性的，导致活性和非活性亚基的异二聚体形成，并且似乎不会引起大规模的结构变化。我们的结果证明了来自肠系膜明串珠菌的Glu-6-PDH对体内已知存在的氧化系统修饰的固有敏感性。对Fe(2+)催化的Glu-6-PDH氧化的生理意义的评估有待将这些研究扩展到已知随着年龄增长会积累活性较低或无活性形式的该酶的哺乳动物来源。
