Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel.
The National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel.
J Mol Biol. 2018 Mar 2;430(5):668-681. doi: 10.1016/j.jmb.2018.01.009. Epub 2018 Feb 2.
The Pup-proteasome system (PPS) is a prokaryotic tagging and degradation system analogous in function to the ubiquitin-proteasome system (UPS). Like ubiquitin, Pup is conjugated to proteins, tagging them for proteasomal degradation. However, in the PPS, a single Pup-ligase, PafA, conjugates Pup to a wide variety of proteins. PafA couples ATP hydrolysis to formation of an isopeptide bond between Pup and a protein lysine via a mechanism similar to that used by glutamine synthetase (GS) to generate glutamine from ammonia and glutamate. GS can also transfer the glutamyl moiety from glutamine to a hydroxyl amine in an ATP-independent manner. Recently, the ability of PafA to transfer Pup from one protein to another was demonstrated. Here, we report that such PafA activity mechanistically resembles the transferase activity of GS. Both PafA and GS transferase activities are ATP-independent and proceed in two catalytic steps. In the first step catalyzed by PafA, an inorganic phosphate is used by the enzyme to depupylate a Pup donor, while forming an acyl phosphate Pup intermediate. The second step consists of Pup conjugation to the new protein, alongside the release of an inorganic phosphate. Detailed experimental analysis, combined with kinetic modeling of PafA transferase activity, allowed us to correctly predict the kinetics and magnitude of Pup transfer between two targets, and analyze the effects of their affinity to PafA on the efficiency of transfer. By deciphering the mechanism of the PafA transferase reaction in kinetic detail, this work provides in-depth mechanistic understanding of PafA, a key PPS enzyme.
Pup-蛋白酶体系统 (PPS) 是一种类似于泛素-蛋白酶体系统 (UPS) 的功能的原核标记和降解系统。与泛素一样，Pup 与蛋白质结合，将其标记为蛋白酶体降解。然而，在 PPS 中，单个 Pup 连接酶 PafA 将 Pup 连接到各种蛋白质上。PafA 通过类似于谷氨酰胺合成酶 (GS) 将氨和谷氨酸转化为谷氨酰胺的机制，将 ATP 水解与 Pup 和蛋白质赖氨酸之间的异肽键形成偶联。GS 还可以以不依赖 ATP 的方式将谷氨酰基部分从谷氨酰胺转移到羟胺上。最近，已经证明了 PafA 将 Pup 从一种蛋白质转移到另一种蛋白质的能力。在这里，我们报告说，这种 PafA 活性在机制上类似于 GS 的转移酶活性。PafA 和 GS 转移酶活性都是不依赖于 ATP 的，并且经过两个催化步骤进行。在 PafA 催化的第一步中，酶使用无机磷酸盐去磷酸化 Pup 供体，同时形成酰基磷酸 Pup 中间物。第二步包括 Pup 与新蛋白质的结合，同时释放无机磷酸盐。详细的实验分析，结合对 PafA 转移酶活性的动力学建模，使我们能够正确预测两个靶标之间的 Pup 转移的动力学和幅度，并分析它们与 PafA 的亲和力对转移效率的影响。通过详细阐明 PafA 转移酶反应的机制，这项工作深入了解了 PPS 关键酶 PafA 的机制。
