E3泛素连接酶，作为泛素化修饰过程中数量和种类最多、催化机制最为复杂的一类酶，一般决定着底物或位点的选择性以及泛素修饰的链类型，调控着细胞内几乎所有的生理过程。然而E3酶的催化过程瞬时动态，蛋白间互作微弱，尤其是催化中间体高度不稳定极难捕获，为解析酶的催化机制带来了挑战；此外获取均质的携带修饰的底物样品也是E3酶活性探究的制约因素。蛋白化学合成手段能够调控氨基酸的序列和结构，可以用于制备均质的修饰蛋白，以及构建催化中间体的稳定模拟物，为E3酶的活性和机制研究提供有力的技术支撑。本论文首先聚焦于反应条件温和、催化速率快的化学酶法，为克服经典酶法存在的可逆性和识别序列依赖性等固有缺陷，发展了底物硫酯协助的酶法连接新策略；然后利用该策略高效制备修饰底物和催化状态的交联缀合物，从而助力E3酶催化活性和机制研究。 鉴于现有酶法难以满足修饰底物的合成需求，我们发展了高效的底物硫酯协助的Sortase（SrtA）酶法连接新策略。新策略增强了酶法的合成能力，不仅克服了传统方法的反应可逆性，而且拓展了酶对识别序列的容忍性，可以用于高效制备带有修饰的、且保留天然氨基酸序列的蛋白。我们利用该策略分别合成了携带单、双和三甲基化修饰的保留天然序列的组蛋白H3，对比了E3酶UHRF1对不同甲基化状态的底物的活性差异，探究了底物甲基化对E3酶活性的影响。 利用发展的酶法合成新策略和多功能小分子CAET介导的连接反应，制备了E3酶CRL1在底物上延伸二泛素时形成的E2-泛素（Ub）-底物催化中间体的稳定模拟物，并结合冷冻电镜、质谱交联等技术和突变体的生化活性实验，搭建了CRL1利用E2酶CDC34催化底物P27生成K48特异性泛素链的机制模型。我们发现受体蛋白SKP2与Ub间的互作扮演着重要功能，不仅有助于复合物形成闭环构象，而且使得受体Ub的K48位点朝向CDC34的活性中心。这可以解释K48泛素链生成的特异性，以及在底物上装载第一个Ub时缓慢的速率。CDC34-Ub部分具有一定的动态性，这可能是快速生成泛素链的基础。NEDD8的密度缺失，表明其可能并不参与稳定复合物的构象。此外，由CRL往底物上延伸四泛素时的电镜密度也发现了SKP2与Ub的互作，表明该催化机制在泛素链延伸过程中的保守性。 综上所述，本文通过发展高效的基于酶法的蛋白质半合成策略，助力研究E3酶UHRF1的活性影响因素，和CRL家族E3酶在底物上延伸泛素链的催化机制。

E3 ligases, with the largest number and variety and the most complex catalytic mechanism in the ubiquitin system, generally determine the selectivity of the modified substrate or ubiquitination site and the type of ubiquitin chains, involving in almost all physiological processes. However, E3 catalysis is dynamic, enzyme-substrate interactions are transient with low affinity, and intermediates in particular are highly unstable and fleeting, which hinders structural insights into the E3-catalyzed ubiquitin (Ub) transfer mechanism. In addition, obtaining homogeneous samples of modified substrates is also a limiting factor for the exploration of E3 activity. Chemical protein synthesis methods can tune the sequence and structure of amino acids, and can be used to prepare homogeneous modified proteins and to construct stable mimics of catalytic intermediates, providing strong technical support for the activity and mechanism research of E3. This thesis first focused on the chemoenzymatic semi-synthetic strategy with mild reaction conditions and fast catalytic rate, and a new strategy for substrate thioester-assisted enzymatic ligation was developed in order to overcome the inherent defects of canonical enzymatic methods such as reversibility and recognition sequence dependence. Then this strategy was used to efficiently prepare modified substrates and stable mimics of intermediates, thereby facilitating E3 catalytic activity and mechanism studies. In view of the difficulty of meeting the needs of the synthesis of modified substrates by the existing enzymatic methods, we developed a new strategy for the efficient thioester-assisted SrtA enzymatic ligation of substrates. The strategy augmented the enzymatic synthetic capability, not only overcoming the reaction reversibility of traditional methods, but also expanding the tolerance of enzymes to recognition sequences, and could be used to efficiently prepare proteins with modifications and retain native amino acid sequences. We used this strategy to synthesize histone H3 with native sequence with mono-, di- and tri-methylation modifications respectively, and compared the activity differences of E3 enzyme UHRF1 on substrates with different methylation states, and explored the effect of substrate methylation on E3 activity. Using the developed novel chemoenzymatic semi-synthetic strategy and the multifunctional molecule CAET-mediated ligation, a stable mimic of the E2-Ub-substrate intermediate was prepared when E3 enzyme CRL1 relied on the E2 enzyme CDC34 to extend ubiquitin chains. Combined with cryo-electron microscopy, chemical crosslinking coupled with mass spectrometry and other techniques, as well as the determination of enzyme activity, a mechanism model of CRL1 using CDC34 to catalyze the substrate P27 to generate K48-specific ubiquitin chains was constructed. We found that the interaction of receptor protein SKP2 with Ub played an important role, not only helping the complex to form a closed-loop conformation, but also orienting the K48 site of receptor Ub toward the catalytic site of CDC34. This could explain the specificity of K48 ubiquitin chain generation and the slow loading of the first Ub onto the substrate. The CDC34-Ub region had certain dynamics, which might be the basis for the rapid generation of ubiquitin chains. The density of NEDD8 was absent, suggesting that it might not be involved in stabilizing the conformation of the complex. In addition, the interaction between SKP2 and Ub was also found in the electron microscope density when the tetraubiquitin chain was extended on the substrate, suggesting that the catalytic mechanism was conserved during ubiquitin chain elongation. In summary, this thesis helps to study the factors affecting the activity of E3 enzyme UHRF1 and the catalytic mechanism of CRL family E3 enzymes extending ubiquitin chains on substrates by developing an efficient enzymatic-based protein semi-synthesis strategy.