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 found that tethering can enhance reaction rates in a flexible tethered kinase system and that the magnitude of the effect is sensitive to the structure of the tether. The largest effective molarity we obtained was ∼0.08 μM, which is much lower than the effects observed in small molecule model systems and other tethered protein reactions. We further demonstrated that the tethered intramolecular reaction only makes a significant contribution to the 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 engineer synthetic networks.