School of Engineering and Science, Jacobs University Bremen, Campus Ring 1, 28759 Bremen, Germany.
Chemistry. 2012 Mar 19;18(12):3521-8. doi: 10.1002/chem.201103397. Epub 2012 Feb 24.
The demand for practical and convenient enzyme assays for histone lysine methyltransferases (HKMTs) emerges along with the rapid development of this young class of enzymes. A supramolecular reporter pair composed of p-sulfonatocalix[4]arene (CX4) and the fluorescent dye lucigenin (LCG) has been used to monitor enzymatic trimethylation of lysine residues in peptide substrates. The assay affords a switch-ON fluorescence response and operates in a continuous, real-time, and label-free fashion. The underlying working principle relies on the higher affinity of the macrocycle towards the trimethylated product of the enzymatic reaction as compared to the substrate, which allows the assay to be carried out in the product-selective mode. The final product incorporates a trimethylammonium moiety, a known high-affinity binding motif for CX4. Two substrates corresponding to the H3 N-terminal tail, namely, S2 (RTKQTARKSTGGKAP) and S6 (QTARKSTGGS), were selected as model compounds for methylation with the Neurospora crassa Dim-5 enzyme and investigated by the newly developed supramolecular tandem HKMTs assay. Only the longer substrate S2 underwent methylation in solution. The potential of the assay for inhibitor screening was demonstrated by means of inhibition studies with 1,10-phenanthroline to afford an inhibition constant of (70±20) μM.
随着这一新兴酶类的快速发展，人们对组蛋白赖氨酸甲基转移酶（HKMTs）实用且便捷的酶活性检测方法的需求也与日俱增。我们构建了一个超分子报告基团对，由对磺酸钠杯[4]芳烃（CX4）和荧光染料虫荧光素（LCG）组成，用于监测肽底物中赖氨酸残基的酶三甲基化。该检测方法提供了一种“开启”型荧光响应，并且可以连续、实时、无标记地进行操作。其工作原理是基于酶反应的三甲基化产物与底物相比，对大环具有更高的亲和力，这使得该检测方法可以在产物选择性模式下进行。最终产物包含一个三甲基铵部分，这是已知与 CX4 具有高亲和力的结合基序。我们选择了两个对应于 H3 N 末端尾巴的底物，即 S2（RTKQTARKSTGGKAP）和 S6（QTARKSTGGS），作为Neurospora crassa Dim-5 酶甲基化的模型化合物，并通过新开发的超分子串联 HKMTs 检测方法进行了研究。只有较长的底物 S2 在溶液中发生了甲基化。通过使用 1,10-菲啰啉进行抑制研究，证明了该检测方法在抑制剂筛选方面的潜力，得到了（70±20）μM 的抑制常数。
