Reiss Y, Heller H, Hershko A
Unit of Biochemistry, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa.
J Biol Chem. 1989 Jun 25;264(18):10378-83.
It was found previously that the enzyme ubiquitin-protein ligase (E3) contains specific protein substrate binding sites that are responsible for the selection of proteins for degradation by the ubiquitin system. In the present study, we have tried to gain more insight into the mode of action of E3 by the characterization of other binding sites of this enzyme. Following the ligation of ubiquitin to 125I-lysozyme, the conjugates produced are very tightly bound to E3, as indicated by size analysis on glycerol density gradient centrifugation. The strong binding of ubiquitin-protein conjugates to the enzyme may account for the apparently processive addition of multiple molecules of ubiquitin to the protein substrate. Both the protein substrate moiety and the ubiquitin moiety participate in the interaction of ubiquitin-protein conjugates with E3, as indicated by competition with specific agents and by the comparison of the binding of ubiquitin-conjugated protein to that of free protein. In addition to the binding of its substrates and products, E3 also appears to interact with some of the enzymes with which it acts in concert. When E3 is incubated with the ubiquitin-carrier protein E2, a complex is formed between the two enzymes as analyzed on glycerol gradients. The formation of an E2.E3 complex may facilitate the transfer of activated ubiquitin from E2 to the protein substrate bound to the ligase.
先前发现，泛素 - 蛋白连接酶（E3）含有特定的蛋白质底物结合位点，这些位点负责选择要被泛素系统降解的蛋白质。在本研究中，我们试图通过对该酶的其他结合位点进行表征，来更深入地了解E3的作用模式。将泛素与¹²⁵I - 溶菌酶连接后，通过甘油密度梯度离心进行大小分析表明，产生的缀合物与E3紧密结合。泛素 - 蛋白缀合物与该酶的强结合可能解释了泛素分子向蛋白质底物上明显的持续添加过程。蛋白质底物部分和泛素部分都参与了泛素 - 蛋白缀合物与E3的相互作用，这通过与特定试剂的竞争以及泛素缀合蛋白与游离蛋白结合的比较得以表明。除了结合其底物和产物外，E3似乎还与一些与其协同作用的酶相互作用。当E3与泛素载体蛋白E2一起孵育时，如在甘油梯度上分析的那样，两种酶之间形成了复合物。E2·E3复合物的形成可能有助于将活化的泛素从E2转移到与连接酶结合的蛋白质底物上。
