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首页> 外文期刊>Biochemistry >Caught before Released: Structural Mapping of the Reaction Trajectory for the Sofosbuvir Activating Enzyme, Human Histidine Triad Nucleotide Binding Protein 1 (hHint1)
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Caught before Released: Structural Mapping of the Reaction Trajectory for the Sofosbuvir Activating Enzyme, Human Histidine Triad Nucleotide Binding Protein 1 (hHint1)

机译：在释放之前捕获：Sofosbuvir激活酶的反应轨迹的结构映射，人组氨酸三元核苷酸结合蛋白1（Hhint1）

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摘要

Human histidine triad: nucleotide binding protein 1 (hHintl) is classified as an efficient nucleoside phosphoramidase and aryl-adenosine monophosphate hydrolase. Human Hintl has been shown to be essential for the metabolic activation of nucleotide antiviral pronucleotides (i.e., proTides), such as the FDA approved hepatitis C drug, sofosbuvir. The active site of hHintl comprises an ensemble of strictly, conserved histidines, including nucleophilic His112. To structurally investigate the mechanism of hHintl catalysis, we have designed and prepared nucleoside thiophosphoramidate substrates that are able to capture the transiently formed nudeotidylated-His112 intermediate (E*) using time-dependent crystallography. Utilizing a catalytically inactive hHintl His112Asn enzyme variant and wild-type enzyme, the enzyme substrate (ES1) and product (EP2) complexes were also cocrystallized, respectively, thus providing a structural map of the reaction trajectory. On the basis of these observations and the mechanistic necessity of proton transfers, proton inventory studies were carried out. Although we cannot completely exclude the possibility of more than one proton in flight, the results of these studies were consistent with the transfer of a single proton during the formation of the intermediate. Interestingly, structural analysis revealed that the critical proton transfers required for intermediate formation and hydrolysis may be mediated by a conserved active site water channel. Taken together, our results provide mechanistic insights underpinning histidine nucleophilic catalysis in general and hHintl catalysis, in particular, thus aiding the design of future proTides and the elucidation of the natural function of the Hint family of enzymes.
机译：人组氨酸三合会：核苷酸结合蛋白1（HHINT1）被分类为一种有效的核苷磷酰胺酶和芳基 - 腺苷单磷酸酶。人文Hintl已被证明对于核苷酸抗病毒激活核苷酸的代谢活化至关重要，例如FDA批准的丙型肝炎药物，Sofosbuvir。 HhIntl的活性位点包括严格，保守的组氨酸的集合，包括HycoOphilic His112。为了在结构上研究Hhintl催化的机制，我们设计了使用时间依赖性晶体学的核苷硫代磷酸酯基底基材，其能够捕获瞬时形成的官方化的官方化蛋白质化 - His112中间体（E *）。利用催化活性Hhintl HHINTL HES112ASN酶变体和野生型酶，酶底物（ES1）和产物（EP2）复合物也分别是共聚的，从而提供反应轨迹的结构图。在这些观察结果和质子转移的机械必需品的基础上，进行了质子库存研究。虽然我们不能完全排除飞行中不止一个质子的可能性，但这些研究的结果与在中间体形成过程中的单个质子的转移一致。有趣的是，结构分析显示中间形成和水解所需的关键质子转移可以由保守的活性位点水通道介导。我们的结果占据了一般和Hhintl催化的机械洞察力，特别是催化，从而促进了未来刺激的设计以及阐明了拟合酶的自然功能的阐明。

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  • 来源
    《Biochemistry》 |2017年第28期|共12页
  • 作者

    Shah Rachit; Maize Kimberly M.; Zhou Xin; Finzel Barry C.; Wagner Carston R.;

  • 作者单位

    Univ Minnesota Dept Med Chem 2231 6th St SE 2-141 CCRB Minneapolis MN 55455 USA;

    Univ Minnesota Dept Med Chem 2231 6th St SE 2-141 CCRB Minneapolis MN 55455 USA;

    Univ Minnesota Dept Med Chem 2231 6th St SE 2-141 CCRB Minneapolis MN 55455 USA;

    Univ Minnesota Dept Med Chem 2231 6th St SE 2-141 CCRB Minneapolis MN 55455 USA;

    Univ Minnesota Dept Med Chem 2231 6th St SE 2-141 CCRB Minneapolis MN 55455 USA;

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  • 正文语种 eng
  • 中图分类 生物化学;
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