Selective inactivation of α-ketoglutarate dehydrogenase and pyruvate dehydrogenase:: Reaction of lipoic acid with 4-hydroxy-2-nonenal
Selective inactivation of α-ketoglutarate dehydrogenase and pyruvate dehydrogenase:: Reaction of lipoic acid with 4-hydroxy-2-nonenal
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DOI：
10.1021/bi981512h
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发表时间：
1998-11-10
期刊：
影响因子：
2.9
通讯作者：
Szweda, LI
中科院分区：
文献类型：
--
作者：
Humphries, KM;Szweda, LI
先前的研究已经证实，4-羟基-2-壬烯醛(HNE)是一种高度毒性的脂质过氧化产物，是一种有效的线粒体呼吸抑制物质。HNE通过抑制α-酮戊二酸脱氢酶(KGDH)对呼吸起作用。由于KGDH在新陈代谢中的核心作用，以及越来越多的证据表明，自由基导致了与多种疾病相关的线粒体功能障碍，因此进一步表征其抑制机制是非常有意义的。在本研究中，HNE处理大鼠心脏线粒体，导致KGDH和丙酮酸脱氢酶(PDH)的选择性抑制，而其他NADH连接的脱氢酶和电子链复合体不受影响。KGDH和PDH在结构和催化上是相似的多酶复合体，表明了一种共同的抑制模式。为确定其抑制作用的机制，我们检测了HNE对纯化的KGDH和PDH的影响。这些研究表明，在底物的存在下，HNE的失活作用大大增强，底物减少了硫辛酸的硫原子，这些硫原子共价结合到KGDH和PDH的E2亚基上。此外，HNE引起的酶活性丧失与硫辛酸巯基可利用性的降低密切相关。抗硫辛酸抗体的使用表明，HNE修饰了纯化的酶制剂和线粒体中的硫辛酸，并且这种修饰依赖于底物的存在。因此，这些结果确定了一种潜在的机制，即自由基的产生和随后的脂质过氧化导致KGDH和PDH的特异性修饰，并抑制NADH连接的线粒体呼吸。
Previous research has established that 4-hydroxy-2-nonenal (HNE), a highly toxic product of lipid peroxidation, is a potent inhibitor of mitochondrial respiration. HNE exerts its effects on respiration by inhibiting alpha-ketoglutarate dehydrogenase (KGDH). Because of the central role of KGDH in metabolism and emerging evidence that free radicals contribute to mitochondrial dysfunction associated with numerous diseases, it is of great interest to further characterize the mechanism of inhibition. In the present study, treatment of rat heart mitochondria with HNE resulted in the selective inhibition of KGDH and pyruvate dehydrogenase (PDH), while other NADH-linked dehydrogenases and electron chain complexes were unaffected. KGDH and PDH are structurally and catalytically similar multienzyme complexes, suggesting a common mode of inhibition. To determine the mechanism of inhibition, the effects of HNE on purified KGDH and PDH were examined. These studies revealed that inactivation by HNE was greatly enhanced in the presence of substrates that reduce the sulfur atoms of lipoic acid covalently bound to the E2 subunits of KGDH and PDH. In addition, loss of enzyme activity induced by HNE correlated closely with a decrease in the availability of lipoic acid sulfhydryl groups. Use of anti-lipoic acid antibodies indicated that HNE modified lipoic acid in both purified enzyme preparations and mitochondria and that this modification was dependent upon the presence of substrates. These results therefore identify a potential mechanism whereby free radical production and subsequent lipid peroxidation lead to specific modification of KGDH and PDH and inhibition of NADH-linked mitochondrial respiration.