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Bioactivation of cytotoxic 4-thiaalkanoyl-CoA thioesters by acyl-CoA dehydrogenase and enoyl-CoA hydratase.

机译：酰基辅酶A脱氢酶和烯酰基辅酶A水合酶对细胞毒性的4-硫代烷酰基辅酶A硫酯的生物激活作用。

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摘要

The interaction of 4-thiaacyl-CoA derivatives, modeled from well-studied cytotoxic cysteine-S-conjugates, is examined with the pig kidney medium-chain acyl-CoA dehydrogenase (MCAD). Both competing pathways for the bioactivation of 4--thiaacyl-CoA thioesters are initiated by enzyme-catalyzed a -proton abstraction. Normal b -oxidation of the substrate by MCAD and subsequent hydration by enoyl-CoA hydratase ultimately produces malonyl-semialdehyde-CoA and a thiolate fragment. However, activated 4-thiaacyl-CoA thioesters undergo direct b -elimination of the thiolate fragment with formation of acryloyl-CoA. In several cases, the thiolate produced from b -elimination yields an electrophilic species which inactivates the medium-chain acyl-CoA dehydrogenase.;Inactivation of the recombinant human enzyme (hMCAD) is explored with DCTH-CoA (5,6-dichloro-4-thia-5-hexenoyl-CoA), which produces acryloyl-CoA and 1,2-dichloroethenethiolate upon elimination. MALDI-TOF analysis of DCTH-CoA-treated hMCAD indicates a native mass increase of approximately 859 amu. Although tryptic digest analysis of the modified enzyme suggests the involvement of the catalytic base containing peptide, the adduct is highly unstable and is not identifiable by conventional protein techniques. Preliminary X-ray crystallographic analysis of pig kidney MCAD complexed with DCTH-CoA indicates modification of both the catalytic base, Glu376, and a second active site residue, Glu99. Despite initial proposals that eliminated acryloyl-CoA does not participate in enzyme inactivation, the X-ray structure with DCTH-CoA suggests otherwise. A mechanism involving participation of both elimination products is proposed.;Although both oxidation and elimination pathways for the bioactivation of DCTH-CoA are initiated by the acyl-CoA dehydrogenase, there is no evidence that the involvement of the dehydrogenase is obligatory. Research presented here suggests an alternate route for the bioactivation of cytotoxic 4-thia fatty acids. Thus, the rat liver enoyl-CoA hydratase can catalyze the b -elimination of thiolate fragments from suitably activated 4-thiaacyl-CoA derivatives. In particular, elimination of DCTFTH-CoA (5,6-dichloro-7,7,7-trifluoro-4-thia-5-heptenoyl-CoA) results in the rapid and irreversible inactivation of the enoyl-CoA hydratase. Despite impairment of its normal catalytic function, DCTFTH-CoA-modified enzyme continues to generate and release more electrophilic species from DCTFTH-CoA beyond that necessary for complete inactivation. Although an upper limit to this event is imposed by cross-linking or excessive modification of enzyme subunits, protection of the hydratase by glutathione or proteins may allow the sustained elimination of potentially cytotoxic metabolites in vivo.
机译：用猪肾脏中链酰基辅酶A脱氢酶（MCAD）检查了由研究充分的细胞毒性半胱氨酸-S-缀合物模拟的4-硫酰基辅酶A衍生物的相互作用。通过酶催化的质子提取来引发4-硫代酰基-CoA硫酯生物活化的两种竞争途径。 MCAD对底物进行正常的b氧化作用，随后通过烯酰辅酶A水合酶进行水合作用，最终产生丙二酰半醛辅酶A和硫醇盐片段。然而，活化的4-硫代酰基-CoA硫酯经历硫醇盐片段的直接β-消除，并形成丙烯酰基-CoA。在某些情况下，由b-消除产生的硫醇盐会产生一种亲电子物质，该物质可以使中链酰基CoA脱氢酶失活。;重组人酶（hMCAD）的失活是用DCTH-CoA（5,6-dichloro-4 -thia-5-heexenoyl-CoA），在消除时会生成丙烯酰基-CoA和1,2-二氯乙烯硫醇盐。 DCTH-CoA处理的hMCAD的MALDI-TOF分析表明，天然质量增加了约859 amu。尽管对修饰酶的胰蛋白酶消化分析表明含有催化碱基的肽的参与，但该加合物高度不稳定并且不能通过常规蛋白质技术鉴定。猪肾脏MCAD与DCTH-CoA复合的初步X射线晶体学分析表明，修饰了催化碱基Glu376和第二个活性位点残基Glu99。尽管最初的建议是消除丙烯酰辅酶A不参与酶的灭活，但具有DCTH-CoA的X射线结构却表明并非如此。提出了涉及两种消除产物参与的机制。尽管DCTH-CoA的生物活化的氧化和消除途径均由酰基-CoA脱氢酶引发，但没有证据表明脱氢酶的参与是强制性的。这里提出的研究提出了一种细胞毒性的4-硫代脂肪酸生物激活的替代途径。因此，大鼠肝烯酰-CoA水合酶可以催化来自适当活化的4-硫代酰基-CoA衍生物的硫醇盐片段的β-消除。特别地，消除DCTFTH-CoA（5,6-二氯-7,7,7-三氟-4-硫杂-5-庚烯酰-CoA）导致烯酰-CoA水合酶快速且不可逆地失活。尽管损害了其正常的催化功能，但DCTFTH-CoA修饰的酶继续产生并从DCTFTH-CoA中释放出更多的亲电物种，超出了完全灭活所必需的亲电子物种。尽管此事件的上限是酶亚基的交联或过度修饰所强加的，但谷胱甘肽或蛋白质对水合酶的保护可允许在体内持续消除潜在的细胞毒性代谢产物。

著录项

  • 作者

    Baker-Malcolm, Jennifer Fay.;

  • 作者单位

    University of Delaware.;

  • 授予单位 University of Delaware.;
  • 学科 Biochemistry.;Toxicology.
  • 学位 Ph.D.
  • 年度 1999
  • 页码 262 p.
  • 总页数 262
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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