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首页> 外文期刊>Biochemistry >Pinpoint Chemical Modification of Asp160 in the 49 kDa Subunit of Bovine Mitochondrial Complex I via a Combination of Ligand-Directed Tosyl Chemistry and Click Chemistry
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Pinpoint Chemical Modification of Asp160 in the 49 kDa Subunit of Bovine Mitochondrial Complex I via a Combination of Ligand-Directed Tosyl Chemistry and Click Chemistry

机译：通过配体导的甲硅化学组合，点击牛线粒体复合物I的49kDa亚基的Asp160的化学改性，然后点击化学

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摘要

Through a ligand-directed tosyl (LDT) chemistry strategy using the synthetic acetogenin ligand AL1, we succeeded in the pinpoint alkynylation (-C=CH) of Asp160 in the 49 kDa subunit of bovine complex I, which may be located in the inner part of the putative quinone binding cavity of the enzyme [Masuya, T., et al. (2014) Biochemistry, 53, 2307-2317]. This study provided a promising technique for diverse chemical modifications of complex I. To further improve this technique for its adaptation to intact complex I, we here synthesized the new acetogenin ligand AL2, possessing an azido (-N3) group in place of the terminal alkyne in AL1, and attempted the pinpoint azidation of complex I in bovine heart submitochondrial particles. Careful proteomic analyses revealed that, just as in the case of AL1, azidation occurred at 49 kDa Asp160 with a reaction yield of similar to 50%, verifying the high site specificity of our LDT chemistry using acetogenin ligands. This finding prompted us to speculate that a reactivity of the azido group incorporated into Asp160 (Asp160-N-3) against externally added chemicals can be employed to characterize the structural features of the quinone/inhibitor binding cavity. Consequently, we used a ring-strained cycloalkyne possessing a rhodamine fluorophore (TAMRA-DIBO), which can covalently attach to an azido group via so-called click chemistry without Cu1+ catalysis, as the reaction partner of Asp160-N-3. We found that bulky TAMRA-DIBO is capable of reacting directly with Asp160-N-3 in intact complex I. Unexpectedly, the presence of an excess amount of short-chain ubiquinones as well as some strong inhibitors (e.g., quinazoline and fenpyroximate) did not interfere with the reaction between TAMRA-DIBO and Asp160-N-3; nevertheless, bullatacin, a member of the natural acetogenins, markedly interfered with this reaction. Taking the marked bulkiness of TAMRA-DIBO into consideration, it appears to be difficult to reconcile these results with the proposal that only a narrow entry point accessing to the quinone/inhibitor binding cavity exists in complex I [Baradaran, R., et al. (2013) Nature, 494, 443-448]; rather, they suggest that there may be another access path for TAMRA-DIBO to the cavity.
机译：通过使用合成醋酸乙酰氨基配体AL1的配体定向的甲硅烷基（LDT）化学策略，在牛络合物I的49kDa亚基中，在ASP160的Pinpoint alkynylation（-C = CH）中，其可以位于内部酶的推定醌结合腔[Masuya，T.等人。 （2014）生物化学，53,2307-2317]。本研究提供了一种有希望的复杂化学修饰的有希望的技术。为了进一步改善其适应完整复合物I的技术，我们在这里合成了具有Azido（-N3）组的新的丙酮糖原配体Al2代替终端炔烃在Al1，并尝试在牛心脏提交粒子中针对复合物I的精确响铃。仔细的蛋白质组学分析显示，与Al1的情况一样，在49kDa Asp160的情况下发生令人吻合，其反应产率类似于50％，验证我们LDT化学的高位特异性使用丙酮素配体。该发现促使我们推测掺入Asp160（Asp160-N-3）的Azido基团的反应性可用于表征醌/抑制剂结合腔的结构特征。因此，我们使用具有罗丹明荧光团（Tamra-Dibo）的环状环晶，其可以通过所谓的咔哒化学将其与无CU1 +催化的所谓的Click Chatony共价连接到Asp160-N-3的反应伴侣。我们发现庞大的Tamra-dibo能够直接与Asp160-N-3在完整的复合物I中直接反应。出乎意料地，存在过量的短链泛醌以及一些强抑制剂（例如，喹唑啉和困倦）的存在不干扰Tamra-Dibo和Asp160-N-3之间的反应;然而，Bullatacin是天然丙酮苷的成员，显着干扰了这种反应。考虑到Tamra-Dibo的标记弱势，似乎难以调和这些结果，该提案只有在复合物I中存在进入醌/抑制剂结合腔的狭窄入口点[巴拉多兰，R.等人。 （2013）自然，494,443-448];相反，他们建议可以存在Tamra-dibo到腔的另一个进入路径。

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  • 来源
    《Biochemistry》 |2014年第49期|共8页
  • 作者

    Masuya Takahiro; Mural Masatoshi; Morisaka Hironobu; Miyoshi Hideto;

  • 作者单位

    Kyoto Univ Grad Sch Agr Div Appl Life Sci Sakyo Ku Kyoto 6068502 Japan;

    Kyoto Univ Grad Sch Agr Div Appl Life Sci Sakyo Ku Kyoto 6068502 Japan;

    Kyoto Univ Grad Sch Agr Div Appl Life Sci Sakyo Ku Kyoto 6068502 Japan;

    Kyoto Univ Grad Sch Agr Div Appl Life Sci Sakyo Ku Kyoto 6068502 Japan;

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  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 生物化学;
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