Specificity of the anaphase-promoting complex: a single-molecule study.

Biological processes require specific enzymatic reactions, paradoxically involving short recognition sequences. As an example, cell-cycle timing depends on a sequence of ubiquitylation events mediated by the anaphase-promoting complex (APC) based on short redundant motifs. To understand the origin of specificity, we designed single-molecule fluorescence assays that capture transient ubiquitylation reactions. We find that the APC-mediated ubiquitylation involves a highly processive initial reaction on the substrate, followed by multiple encounters and reactions at a slower rate. The initial ubiquitylation greatly enhances the substrate's binding affinity in subsequent reactions, by both increasing the on-rate and decreasing the off-rate. We postulate that these cycles of positive feedback enable high specificity for substrates with short recognition motifs in a complex cellular environment.