Lys ubiquitination is catalysed by E3 ubiquitin ligases and is central to the regulation of protein stability and cell signalling in normal and disease states. There are gaps in our understanding of E3 mechanisms, and here we use protein semisynthesis, chemical rescue, microscale thermophoresis and other biochemical approaches to dissect the role of catalytic base/acid function and conformational interconversion in HECT-domain E3 catalysis. We demonstrate that there is plasticity in the use of the terminal side chain or backbone carboxylate for proton transfer in HECT E3 ubiquitin ligase reactions, with yeast Rsp5 orthologues appearing to be possible evolutionary intermediates. We also show that the HECT-domain ubiquitin covalent intermediate appears to eject the E2 conjugating enzyme, promoting catalytic turnover. These findings provide key mechanistic insights into how protein ubiquitination occurs and provide a framework for understanding E3 functions and regulation.

Lys 泛素化由 E3 泛素连接酶催化，对于正常和疾病状态下蛋白质稳定性和细胞信号传导的调节至关重要。我们对E3机制的理解存在差距，在这里我们利用蛋白质半合成、化学拯救、微尺度热泳等生化方法来剖析催化碱/酸功能和构象互变在HECT域E3催化中的作用。我们证明，在 HECT E3 泛素连接酶反应中使用末端侧链或主链羧酸盐进行质子转移具有可塑性，酵母 Rsp5 直系同源物似乎是可能的进化中间体。我们还表明，HECT 结构域泛素共价中间体似乎会排出 E2 结合酶，从而促进催化周转。这些发现提供了关于蛋白质泛素化如何发生的关键机制见解，并为理解 E3 功能和调节提供了框架。