The Shraga Segal Department of Microbiology, Immunology and Genetics, Ben-Gurion University of the Negev, Be'er Sheva 84105, Israel; The National Institute for Biotechnology in the Negev (NIBN), Ben-Gurion University of the Negev, Be'er Sheva 84105, Israel.
The National Institute for Biotechnology in the Negev (NIBN), Ben-Gurion University of the Negev, Be'er Sheva 84105, Israel; Department of Life Sciences, Ben-Gurion University of the Negev, Be'er Sheva 84105, Israel.
Biochem Biophys Res Commun. 2018 Jul 2;501(4):1029-1033. doi: 10.1016/j.bbrc.2018.05.103. Epub 2018 May 24.
Protein lysine methyltransferases (PKMTs) catalyze the methylation of lysine residues on many different cellular proteins. Despite extensive biochemical and structural studies, focusing on PKMT active site-peptide interactions, little is known regarding how PKMTs recognize globular substrates. To examine whether these enzymes recognize protein substrates through interactions that take place outside of the active site, we have measured SETD6 and SETD7 activity with both protein and peptide RelA substrate. We have utilized the MTase-Glo™ methyltransferase assay to measure the activity of SETD6 and SETD7 with the different RelA substrates and calculated the Michaelis-Menten (MM) parameters. We found an up to ∼12-fold increase in K of the PKMTs activity with RelA peptide relative to the respective full-length protein, emphasizing the significantly higher PKMT-protein interaction affinity. Examination of SETD6 and SETD7 activity toward the same RelA substrates highlight the similarity in substrate recognition for both PKMTs. Our results show that the interaction affinity of SETD6 and SETD7 with RelA is enhanced through interactions that occur outside of the active site leading to higher catalytic efficiency and specificity. These interactions can significantly vary depending on the PKMT and the specific methylation site on RelA. Overall, our results underline that PKMTs can recognize their substrates through docking interactions that occur out of the active site-peptide region for enhancing their activity and specificity in the cellular environment.
蛋白赖氨酸甲基转移酶（PKMTs）催化多种不同细胞蛋白赖氨酸残基的甲基化。尽管对 PKMT 活性位点-肽相互作用进行了广泛的生化和结构研究，但对于 PKMT 如何识别球状底物知之甚少。为了研究这些酶是否通过发生在活性位点之外的相互作用来识别蛋白质底物，我们使用蛋白和肽 RelA 底物来测量 SETD6 和 SETD7 的活性。我们利用 MTase-Glo™甲基转移酶测定法来测量不同 RelA 底物上 SETD6 和 SETD7 的活性，并计算米氏常数（MM）参数。我们发现 PKMT 活性与 RelA 肽的 K 值相对于各自全长蛋白增加了约 12 倍，这强调了 PKMT-蛋白相互作用亲和力显著提高。对 SETD6 和 SETD7 对相同 RelA 底物的活性的研究突出了两种 PKMT 对底物识别的相似性。我们的结果表明，SET6 和 SETD7 与 RelA 的相互作用亲和力通过发生在活性位点之外的相互作用得到增强，从而导致更高的催化效率和特异性。这些相互作用因 PKMT 和 RelA 上特定的甲基化位点而异。总体而言，我们的结果强调，PKMT 可以通过发生在活性位点-肽区域之外的对接相互作用来识别其底物，从而提高其在细胞环境中的活性和特异性。
