Haas A L, Bright P M
Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226.
J Biol Chem. 1988 Sep 15;263(26):13258-67.
The covalent ligation of the 8.6-kDa polypeptide ubiquitin to various cellular target proteins is believed to represent a fundamental regulatory process. In this mechanism, the ATP-coupled activation and subsequent ligation of ubiquitin are catalyzed by separate enzymes (E1 and E3, respectively) functionally linked by ubiquitin carrier protein (E2). Carrier protein has been proposed to constitute a family of isozymes having molecular masses of 14, 17, 20, 24, and 32 kDa whose role is to shuttle activated polypeptide in the form of a high-energy thiol ester intermediate to the carboxyl terminus of ubiquitin. Using a combination of covalent affinity and high performance liquid chromatographic methods, the pututive E2 isozymes have been purified to apparent homogeneity. The E2(14kDa) isozyme resolved into two forms differing in net charge at pH 7.5. All of the E2 isozymes contained only one thiol ester site except for E2(17kDa) and E2(20kDa) which were capable of forming two such adducts per subunit. Thiol ester formation was rapid for the E2 isozymes and required the presence of activating enzyme. In contrast, the reverse reaction of thiol ester transfer from E2 to E1 was kinetically significant for only E2(14kDa), E2(20kDa), and E2(24kDa). The stability of E2(17kDa) and E2(32kDa) to such trapping may reflect a marked shift in binding affinity to E1 upon thiol ester formation. In addition, differential rates for thiol ester formation to each subunit of dimeric E2(14kDa) was also noted. The E2(14kDa) isoforms were approximately 10-fold more active in E3-dependent ubiquitin-protein ligation than either E2(20kDa) or E2(32kDa). Neither E2(17kDa) nor E2(24kDa) supported this reaction. In addition, the thiol ester formed to E2(14kDa) was inherently more reactive since its second order rate constant for the E3-independent transfer of ubiquitin to the small molecular weight nucleophile dithiothreitol was an order of magnitude greater than found for the other isozymes. If these proteins constitute a family of isozymes, they exhibit considerable catalytic diversity.
8.6千道尔顿的多肽泛素与各种细胞靶蛋白的共价连接被认为是一个基本的调节过程。在这个机制中，泛素的ATP偶联激活及随后的连接分别由泛素载体蛋白（E2）功能连接的不同酶（分别为E1和E3）催化。有人提出载体蛋白构成一个同工酶家族，其分子量分别为14、17、20、24和32千道尔顿，其作用是以高能硫酯中间体的形式将活化的多肽转运到泛素的羧基末端。通过共价亲和与高效液相色谱方法相结合，已将假定的E2同工酶纯化至表观均一性。E2（14千道尔顿）同工酶在pH 7.5时可解析为两种净电荷不同的形式。除了E2（17千道尔顿）和E2（20千道尔顿）每个亚基能够形成两个这样的加合物外，所有E2同工酶仅含有一个硫酯位点。硫酯形成对E2同工酶来说很快，并且需要活化酶的存在。相反，硫酯从E2转移到E1的逆反应仅对E2（14千道尔顿）、E2（20千道尔顿）和E2（24千道尔顿）在动力学上有意义。E2（17千道尔顿）和E2（32千道尔顿）对这种捕获的稳定性可能反映了硫酯形成后与E1结合亲和力的显著变化。此外，还注意到二聚体E2（14千道尔顿）每个亚基硫酯形成的速率不同。E2（14千道尔顿）同工型在依赖E3的泛素 - 蛋白质连接中比E2（20千道尔顿）或E2（32千道尔顿）活性高约10倍。E2（17千道尔顿）和E2（24千道尔顿）都不支持此反应。此外，形成于E2（14千道尔顿）的硫酯本质上更具反应性，因为其在不依赖E3的情况下将泛素转移至小分子亲核试剂二硫苏糖醇的二级速率常数比其他同工酶高一个数量级。如果这些蛋白质构成一个同工酶家族，它们表现出相当大的催化多样性。
