Burgos Emmanuel S, Walters Ryan O, Huffman Derek M, Shechter David
Department of Biochemistry , Albert Einstein College of Medicine , 1300 Morris Park Avenue , Bronx , New York 10461 , USA . Email:
Department of Molecular Pharmacology , Albert Einstein College of Medicine , 1300 Morris Park Avenue , Bronx , New York 10461 , USA.
Chem Sci. 2017 Sep 1;8(9):6601-6612. doi: 10.1039/c7sc02830j. Epub 2017 Jul 27.
Methyltransferases use -adenosyl-l-methionine (SAM) to deposit methyl marks. Many of these epigenetic 'writers' are associated with gene regulation. As cancer etiology is highly correlated with misregulated methylation patterns, methyltransferases are emerging therapeutic targets. Successful assignment of methyltransferases' roles within intricate biological networks relies on (1) the access to enzyme mechanistic insights and (2) the efficient screening of chemical probes against these targets. To characterize methyltransferases and , we report a highly-sensitive one-step deaminase-linked continuous assay where the -adenosyl-l-homocysteine (SAH) enzyme-product is rapidly and quantitatively catabolized to -inosyl-l-homocysteine (SIH). To highlight the broad capabilities of this assay, we established enzymatic characteristics of two protein arginine methyltransferases (PRMT5 and PRMT7), a histone-lysine -methyltransferase (DIM-5) and a sarcosine/dimethylglycine -methyltransferase (SDMT). Since the coupling deaminase TM0936 displays robust activity over a broad pH-range we determined the pH dependence of SDMT reaction rates. TM0936 reactions are monitored at 263 nm, so a drawback may arise when methyl acceptor substrates absorb within this UV-range. To overcome this limitation, we used an isosteric fluorescent SAM-analog: -8-aza-adenosyl-l-methionine. Most enzymes tolerated this probe and sustained methyltransfers were efficiently monitored through loss of fluorescence at 360 nm. Unlike discontinuous radioactive- and antibody-based assays, our assay provides a simple, versatile and affordable approach towards the characterization of methyltransferases. Supported by three logs of linear dynamic range, the 1-Step EZ-MTase can detect methylation rates as low as 2 μM h, thus making it possible to quantify low nanomolar concentrations of glycine -methyltransferase within crude biological samples. With '-factors above 0.75, this assay is well suited to high-throughput screening and may promote the identification of novel therapeutics.
甲基转移酶利用S-腺苷-L-甲硫氨酸（SAM）来沉积甲基标记。许多这类表观遗传“书写者”与基因调控相关。由于癌症病因与甲基化模式失调高度相关，甲基转移酶正成为新兴的治疗靶点。要在复杂的生物网络中成功确定甲基转移酶的作用，依赖于（1）获取酶的作用机制见解，以及（2）针对这些靶点高效筛选化学探针。为了表征甲基转移酶 和 ，我们报告了一种高灵敏度的一步脱氨酶连接连续测定法，其中S-腺苷-L-高半胱氨酸（SAH）酶产物能迅速且定量地分解为S-肌苷-L-高半胱氨酸（SIH）。为了突出该测定法的广泛能力，我们确定了两种蛋白质精氨酸甲基转移酶（PRMT5和PRMT7）、一种组蛋白赖氨酸N-甲基转移酶（DIM-5）和一种肌氨酸/二甲基甘氨酸N-甲基转移酶（SDMT）的酶学特性。由于偶联脱氨酶TM0936在较宽的pH范围内都表现出强大活性，我们测定了SDMT反应速率的pH依赖性。TM0936反应在263nm处监测，所以当甲基受体底物在该紫外范围内有吸收时可能会出现问题。为克服这一限制，我们使用了一种等排体荧光SAM类似物：S-8-氮杂腺苷-L-甲硫氨酸。大多数酶都能耐受这种探针，通过360nm处荧光的损失可有效监测持续的甲基转移。与不连续的放射性和基于抗体的测定法不同，我们的测定法为甲基转移酶的表征提供了一种简单、通用且经济实惠的方法。在三个数量级的线性动态范围内得到支持，一步EZ-甲基转移酶测定法能检测低至2μM/h的甲基化速率，从而能够在粗生物样品中定量低纳摩尔浓度的甘氨酸N-甲基转移酶。由于Z'-因子高于0.75，该测定法非常适合高通量筛选，可能会促进新型治疗药物的鉴定。
