Humphries K M, Szweda L I
Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4970, USA.
Biochemistry. 1998 Nov 10;37(45):15835-41. doi: 10.1021/bi981512h.
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.
先前的研究已证实，4-羟基-2-壬烯醛（HNE）作为脂质过氧化的一种高毒性产物，是线粒体呼吸的强效抑制剂。HNE 通过抑制α-酮戊二酸脱氢酶（KGDH）对呼吸作用产生影响。由于KGDH在新陈代谢中起核心作用，且有新证据表明自由基会导致与多种疾病相关的线粒体功能障碍，因此进一步明确抑制机制具有重要意义。在本研究中，用HNE处理大鼠心脏线粒体导致KGDH和丙酮酸脱氢酶（PDH）被选择性抑制，而其他与NADH相关的脱氢酶和电子链复合物未受影响。KGDH和PDH是结构和催化功能相似的多酶复合物，提示存在共同的抑制模式。为确定抑制机制，研究了HNE对纯化的KGDH和PDH的影响。这些研究表明，在能还原与KGDH和PDH的E2亚基共价结合的硫辛酸硫原子的底物存在时，HNE导致的失活作用会大大增强。此外，HNE诱导的酶活性丧失与硫辛酸巯基可用性的降低密切相关。使用抗硫辛酸抗体表明，HNE在纯化的酶制剂和线粒体中均修饰了硫辛酸，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机制，即自由基产生及随后的脂质过氧化导致KGDH和PDH的特异性修饰以及与NADH相关的线粒体呼吸的抑制。
