Structure and mechanism of the ubiquitin activating enzyme

泛素激活酶激活泛素是泛素分子共价连接到靶底物赖氨酸残基侧链上的第一步。这种修饰存在于多种细胞过程中，包括蛋白酶体对目标蛋白的选择性降解。E1催化的泛素活化是一个三步过程，泛素在其C-末端甘氨酸被腺化，然后泛素与必需的半胱氨酸残基共价连接。最后，该酶确保泛素在跨硫酯化反应中转移到几种泛素结合(E2)酶中的一种，而不是泛素样蛋白修饰物(如SUMO或NEDD8)的任何一种E2酶。基于我们的酵母E1酶与结合在腺化活性部位的泛素之间的络合物的晶体结构，这是完整的泛素E1的第一个晶体结构，我们打算出于以下目的进一步研究该酶的性质：(1)我们将研究该酶如何识别同源的E2酶并区分参与相扑或NEDD8转移的E2酶。(2)我们将表征小分子是如何抑制E1酶的，以进一步探讨其催化机制，并作为设计高特异性抑制剂的第一步。(3)我们将确定一个由E1酶、两个结合的泛素分子和一个E2酶组成的四元络合物的晶体结构，以确定伴随着反式硫代酯化的构象变化。

The activation of ubiquitin by the ubiquitin-activating enzyme (E1) represents the initial step in the covalent attachment of ubiquitin molecules to the side chains of lysine residues in target substrates. This modification is found in a multitude of cellular process including the selected degradation of the target protein by the proteasome. The E1-catalyzed activation of ubiquitin is a three-step process in which ubiquitin is adenylated at its C-terminal glycine, followed by the covalent attachment of ubiquitin to an essential cysteine residue. Finally, the enzyme ensures that ubiquitin is transferred in a trans-thioesterification reaction to one of several ubiquitin conjugating (E2) enzymes but not to any of the E2 enzymes of a ubiquitinlike protein modifier such as SUMO or NEDD8. Based on our crystal structure of the complex between the yeast E1 enzyme with ubiquitin bound at the adenylation active site, the first crystal structure of an intact ubiquitin E1, we intend to further characterize this enzyme with following aims in mind: (1) We will study how the enzyme recognizes cognate E2 enzymes and discriminates against E2 enzymes involved in SUMO or NEDD8 transfer. (2) We will characterize how the E1 enzyme is inhibited by small molecules to further probe its catalytic mechanism and as a first step towards the design of highly specific inhibitors. (3) We will determine the crystal structure of a quarternary complex consisting of the E1 enzyme, two bound ubiquitin molecules and an E2 enzyme to determine the conformational changes accompanying trans-thioesterification.