Fingerprinting differential active site constraints of ATPases
Fingerprinting differential active site constraints of ATPases
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DOI：
10.1039/c3sc21916j
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发表时间：
2013-01-01
期刊：
影响因子：
8.4
通讯作者：
Marx, Andreas
中科院分区：
文献类型：
--
作者：
Hacker, Stephan M.;Hardt, Norman;Marx, Andreas
由三磷酸腺苷（ATP）水解提供的自由能是许多细胞过程的核心，因此，利用ATP作为底物的酶的数量几乎是无数的。修饰的ATP类似物是了解ATP酶生物学功能的重要手段。虽然这些酶已经朝着与ATP结合的方向发展，但发现它们的活性位点结构存在很大差异，为了系统地获得不同ATP酶的特定活性位点限制，需要合适的工具。在这里，我们提出了六个新的基于ATP的ATP酶探针的合成在三个不同的位置的核碱基和核糖，分别修改。随后，我们研究了ATP酶粘着斑激酶FAK，泛素激活蛋白UBA 1和驱动蛋白Eg5作为通过不同机制处理ATP的ATP酶的例子。我们发现，对于这些酶中的每一种，ATP中的至少一个位置可以被修饰而不损失酶的接受。然而，所研究的酶之间容许修饰的位置显著不同，允许绘制反应性指纹。引入的ATP类似物可以形成用于设计定制探针的基础，所述定制探针对感兴趣的特定ATP酶具有增加的亲和力和特异性。
The free energy provided by adenosine triphosphate (ATP) hydrolysis is central to many cellular processes and, therefore, the number of enzymes utilizing ATP as a substrate is almost innumerable. Modified analogues of ATP are a valuable means to understand the biological function of ATPases. Although these enzymes have evolved towards binding to ATP, large differences in active site architectures were found. In order to systematically access the specific active site constraints of different ATPases suitable tools are required. Here, we present the synthesis of six new ATP-based ATPase probes modified at three different positions of the nucleobase and the ribose, respectively. Subsequently, we studied the ATPases focal adhesion kinase FAK, the ubiquitin-activating protein UBA1 and the kinesin Eg5 as examples for ATPases that process ATP by different mechanisms. We find that for each of these enzymes at least one position in ATP can be modified without loss of acceptance by the enzyme. However, the positions at which modifications are tolerated significantly differ between the studied enzymes allowing fingerprints to be drawn for reactivity. The introduced ATP analogues may form the basis for the design of tailored probes with increased affinity and specificity for a specific ATPase of interest.