The relationship between effective molarity and affinity governs rate enhancements in tethered kinase-substrate reactions
Scaffold proteins are thought to accelerate protein phosphorylation reactions by tethering kinases and substrates together, but there is little quantitative data on their functional effects. To assess the contribution of tethering to kinase reactivity, we compared intramolecular and intermolecular kinase reactions in a minimal model system. We find that tethering can enhance reaction rates in a flexible tethered kinase system, and the magnitude of the effect is sensitive to the structure of the tether. The largest effective molarity we obtained was [~]0.08 {micro}M, which is much lower than the effects observed in small molecule model systems and tethered protein-ligand interactions. We further demonstrate that the tethered, intramolecular reaction only makes a significant contribution to observed rates when the scaffolded complex assembles at concentrations below the effective molarity. These findings provide a quantitative framework that can be applied to understand endogenous protein scaffolds and to engineer synthetic networks.