Hershko A, Heller H, Eytan E, Reiss Y
J Biol Chem. 1986 Sep 15;261(26):11992-9.
In order to gain insight into the mechanisms that determine the selectivity of the ubiquitin proteolytic pathway, the protein substrate binding site of the ubiquitin-protein ligase system was identified and examined. Previous studies had shown that the ligase system consists of three components: a ubiquitin-activating enzyme (E1), ubiquitin-carrier protein (E2), and a third enzyme, E3, the mode of action of which has not been defined. E3 from rabbit reticulocytes was further purified by a combination of affinity chromatography, hydrophobic chromatography, and gel filtration procedures. A 180-kDa protein was identified as the subunit of E3. Two independent methods indicate that E3 has the protein binding site of the ubiquitin ligase system. These are the chemical cross-linking of 125I-labeled proteins to the E3 subunit and the functional conversion of enzyme-bound labeled proteins to ubiquitin conjugates in pulse-chase experiments. The trapping of E3-bound protein for labeled product formation was allowed by the slow dissociation of E3 X protein complex. The specificity of binding of different proteins to E3, examined by both methods, showed a direct correlation with their susceptibility to degradation by the ubiquitin system. Proteins with free alpha-NH2 groups, which are good substrates, bind better to E3 than corresponding proteins with blocked NH2 termini, which are not substrates. Oxidation of methionine residues to sulfoxide derivatives greatly increases the susceptibility of some proteins to ligation with ubiquitin, with a corresponding increase in their binding to E3. However, a protein derivative which was subjected to both amino group modification and oxidation binds strongly to the enzyme, even though it cannot be ligated to ubiquitin. It thus seems that the substrate binding site of E3 participates in determining the specificity of proteins that enter the ubiquitin pathway of protein degradation.
为深入了解决定泛素蛋白水解途径选择性的机制，对泛素 - 蛋白连接酶系统的蛋白质底物结合位点进行了鉴定和研究。先前的研究表明，连接酶系统由三个组分组成：泛素激活酶（E1）、泛素载体蛋白（E2）以及第三种酶E3，其作用方式尚未明确。通过亲和层析、疏水层析和凝胶过滤程序相结合的方法，对来自兔网织红细胞的E3进行了进一步纯化。一种180 kDa的蛋白质被鉴定为E3的亚基。两种独立的方法表明E3具有泛素连接酶系统的蛋白质结合位点。这两种方法分别是将125I标记的蛋白质与E3亚基进行化学交联，以及在脉冲追踪实验中将酶结合的标记蛋白质功能性转化为泛素缀合物。E3与蛋白质复合物的缓慢解离使得能够捕获与E3结合的蛋白质以形成标记产物。通过这两种方法检测不同蛋白质与E3结合的特异性，结果显示其与它们被泛素系统降解的敏感性直接相关。具有游离α - NH2基团的蛋白质是良好的底物，它们与E3的结合比相应的NH2末端被封闭的蛋白质更好，而后者不是底物。甲硫氨酸残基氧化为亚砜衍生物极大地增加了一些蛋白质与泛素连接的敏感性，同时它们与E3的结合也相应增加。然而，一种同时进行了氨基修饰和氧化的蛋白质衍生物，即使它不能与泛素连接，也能与该酶强烈结合。因此，似乎E3的底物结合位点参与决定进入泛素介导的蛋白质降解途径的蛋白质的特异性。
