SELECTIVE INACTIVATION OF ALPHA-KETOGLUTARATE DEHYDROGENASE AND PYRUVATE-DEHYDROGENASE - REACTION OF LIPOIC ACID WITH 4-HYDROXY-2-NONENAL

Previous research has established that 4-hydroxy-2-nonenal (HNE), a hi ghly toxic product of lipid peroxidation, is a potent inhibitor of mit ochondrial respiration. HNE exerts its effects on respiration by inhib iting alpha-ketoglutarate dehydrogenase (KGDH). Because of the central role of KGDH in metabolism and emerging evidence that free radicals c ontribute to mitochondrial dysfunction associated with numerous diseas es, it is of great interest to further characterize the mechanism of i nhibition. In the present study, treatment of rat heart mitochondria w ith HNE resulted in the selective inhibition of KGDH and pyruvate dehy drogenase (PDH), while other NADH-linked dehydrogenases and electron c hain complexes were unaffected. KGDH and PDH are structurally and cata lytically similar multienzyme complexes, suggesting a common mode of i nhibition. To determine the mechanism of inhibition, the effects of HN E 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 E 2 subunits of KGDH and PDH. In addition, loss of enzyme activity induc ed by HNE correlated closely with a decrease in the availability of li poic acid sulfhydryl groups. Use of anti-lipoic acid antibodies indica ted that HNE modified lipoic acid in both purified enzyme preparations and mitochondria and that this modification was dependent upon the pr esence of substrates. These results therefore identify a potential mec hanism whereby free radical production and subsequent lipid peroxidati on lead to specific modification of KGDH and PDH and inhibition of NAD H-linked mitochondrial respiration.