Fitzsimmons M E, Thorpe C, Anders M W
Department of Pharmacology, School of Medicine and Dentistry, University of Rochester, New York 14642, USA.
Biochemistry. 1995 Apr 4;34(13):4276-86. doi: 10.1021/bi00013a017.
5,6-Dichloro-4-thia-5-hexenoic acid (DCTH) is a potent hepato- and nephrotoxin that induces mitochondrial dysfunction in rat liver and kidney. Previous studies indicate that DCTH undergoes fatty acid beta-oxidation-dependent bioactivation. The objectives of the present experiments were to elaborate the bioactivation mechanism of DCTH and to examine the interaction of the coenzyme A thioester of DCTH (DCTH-CoA) with the medium-chain acyl-CoA dehydrogenase. In the presence of the terminal electron acceptor ferricenium hexafluorophosphate (FcPF6), DCTH-CoA was oxidized by the medium-chain actyl-CoA dehydrogenase to give 5,6-dichloro-4-thia-trans-2,5-hexadienoyl-CoA. Enoyl-CoA hydratase catalyzed the conversion of 5,6-dichloro-4-thia-trans-2,5-hexadienoyl-CoA to 5,6-dichloro-4-thia-3-hydroxy-5-hexenoyl-CoA, which eliminated 1,2-dichloroethenethiol and gave malonyl-CoA semialdehyde as a product. Chloroacetic acid was detected as a terminal product derived from 1,2-dichloroethenethiol. Incubation of DCTH-CoA with the medium-chain acyl-CoA dehydrogenase in the absence of FcPF6 gave 3-hydroxypropionyl-CoA as the major product and resulted in the irreversible inactivation of the enzyme. Under these conditions, DCTH-CoA apparently undergoes a beta-elimination reaction to give 1,2-dichloroethenethiol and acryloyl-CoA, which is hydrated to give 3-hydroxypropionyl-CoA as the terminal product. The beta-elimination product 1,2-dichloroethenethiol may yield reactive intermediates that inactivate the dehydrogenase. Enzyme inactivation was rapid, DCTH-CoA concentration-dependent, and blocked by octanoyl-CoA, but not by glutathione. The medium-chain acyl-CoA dehydrogenase was not inactivated by acryloyl-CoA, and little inactivation was observed in the presence of FcPF6. These results show that DCTH-CoA is bioactivated by the mitochondrial fatty acid beta-oxidation system to reactive intermediates. This bioactivation mechanism may account for the observed toxicity of DCTH in vivo and in vitro.
5,6-二氯-4-硫杂-5-己烯酸（DCTH）是一种强效的肝毒素和肾毒素，可诱导大鼠肝脏和肾脏中的线粒体功能障碍。先前的研究表明，DCTH经历脂肪酸β-氧化依赖性生物活化。本实验的目的是阐述DCTH的生物活化机制，并研究DCTH的辅酶A硫酯（DCTH-CoA）与中链酰基辅酶A脱氢酶的相互作用。在末端电子受体六氟磷酸铁鎓（FcPF6）存在的情况下，DCTH-CoA被中链乙酰辅酶A脱氢酶氧化，生成5,6-二氯-4-硫杂-反式-2,5-己二烯酰辅酶A。烯酰辅酶A水合酶催化5,6-二氯-4-硫杂-反式-2,5-己二烯酰辅酶A转化为5,6-二氯-4-硫杂-3-羟基-5-己烯酰辅酶A，后者消除1,2-二氯乙硫醇并生成丙二酰辅酶A半醛作为产物。检测到氯乙酸是源自1,2-二氯乙硫醇的终产物。在不存在FcPF6的情况下，将DCTH-CoA与中链酰基辅酶A脱氢酶一起孵育，得到3-羟基丙酰辅酶A作为主要产物，并导致该酶不可逆失活。在这些条件下，DCTH-CoA显然经历β-消除反应，生成1,2-二氯乙硫醇和丙烯酰辅酶A，后者水合生成3-羟基丙酰辅酶A作为终产物。β-消除产物1,2-二氯乙硫醇可能产生使脱氢酶失活的反应性中间体。酶失活迅速，呈DCTH-CoA浓度依赖性，并被辛酰辅酶A阻断，但不被谷胱甘肽阻断。中链酰基辅酶A脱氢酶不会被丙烯酰辅酶A失活，并且在FcPF6存在的情况下几乎没有观察到失活现象。这些结果表明，DCTH-CoA被线粒体脂肪酸β-氧化系统生物活化成反应性中间体。这种生物活化机制可能解释了在体内和体外观察到的DCTH的毒性。
