One-pot and sequential organic chemistry on an enzyme surface to tether a fluorescent probe at the proximity of the active site with restoring enzyme activity
One-pot and sequential organic chemistry on an enzyme surface to tether a fluorescent probe at the proximity of the active site with restoring enzyme activity
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DOI：
10.1021/ja057926x
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发表时间：
2006-03-15
影响因子：
15
通讯作者：
Hamachi, I
中科院分区：
文献类型：
--
作者：
Takaoka, Y;Tsutsumi, H;Hamachi, I
已经成功开发出一种新的简单方法，可以将功能分子束缚在酶活性位点附近，而不会损失任何活性。基于亲和标记和随后的腙/肟交换反应，在人碳酸酐酶 II (hCAII) 表面上进行一锅序贯反应。整个步骤的反应在温和的条件下以大于90%的产率进行。酶活性测定表明，亲和配体从hCAII活性位点的释放与氨氧基衍生物的取代同时发生，从而使标记的hCAII的酶活性从完全抑制的状态恢复。与传统的亲和标记方法相比，连续修饰后活性的恢复是非常独特的。肽图谱实验表明，标记反应选择性地针对位于靠近活性位点的蛋白质表面上的 His-3 或 His-4。当使用本发明的连续化学方法连接荧光探针时，工程化的 hCAII 可以充当针对 hCAII 抑制剂的荧光生物传感器。这清楚地表明了该方法的两个优点，即（i）修饰针对活性位点附近，以及（ii）顺序反应重新打开目标酶的活性位点空腔。
A new and simple method to tether a functional molecule at the proximity of the active site of an enzyme has been successfully developed without any activity loss. The one-pot sequential reaction was conducted on a surface of human carbonic anhydrase II (hCAII) based on the affinity labeling and the subsequent hydrazone/oxime exchange reaction. The reaction proceeds in a greater than 90% yield in the overall steps under mild conditions. The enzymatic activity assay demonstrated that the release of the affinity ligand from the active site of hCAII concurrently occurred with the replacement by the aminooxy derivatives, so that it restored the enzymatic activity from the completely suppressed state of the labeled hCAII. Such restoring of the activity upon the sequential modification is quite unique compared to conventional affinity labeling methods. The peptide mapping experiment revealed that the labeling reaction was selectively directed to His-3 or His-4, located on a protein surface proximal to the active site. When the fluorescent probe was tethered using the present sequential chemistry, the engineered hCAII can act as a fluorescent biosensor toward the hCAII inhibitors. This clearly indicates the two advantages of this method, that is (i) the modification is directed to the proximity of the active site and (ii) the sequential reaction re-opens the active site cavity of the target enzyme.