The Role of Activator in Substrate Ubiquitination by the Anaphase-Promoting Complex

The anaphase-promoting complex/cyclosome (APC/C) is a protein-ubiquitin ligase (E3) that initiates chromosome segregation. Like other RING E3s, the APC/C catalyzes ubiquitin transfer from an E2-ubiquitin (E2-Ub) conjugate to a specific protein substrate. APC/C activity requires association with an activator subunit that recruits substrate and might also enhance catalysis, but the biochemical function of the activator is unclear. We dissected activator function using an artificial fusion substrate that binds tightly to the APC/C and is ubiquitinated at a low rate in the absence of activator. Ubiquitination of this substrate was stimulated by activator, due primarily to a dramatic stimulation of E2 sensitivity (Km) and catalytic rate (kcat), which together resulted in a 650-fold stimulation of kcat/Km. Thus, activator is not simply a substrate adaptor but also enhanced catalysis by promoting a more efficient interaction with the E2-Ub. Interestingly, APC/C stimulation by activator required activator interaction with degron sequence motifs on the substrate. We conclude that formation of a complete APC/C-activator-substrate complex leads to a major enhancement of E2 efficiency, providing an unusual substrate-assisted catalytic mechanism that limits efficient ubiquitin transfer to specific substrates