Intrinsically disordered linkers control tethered kinases via effective concentration.

Kinase specificity is crucial to the fidelity of signaling pathways, yet many pathways use the same kinases to achieve widely different effects. Specificity arises in part from the enzymatic domain but also from the physical tethering of kinases to their substrates. Such tethering can occur via protein interaction domains in the kinase or via anchoring and scaffolding proteins and can drastically increase the kinetics of phosphorylation. However, we do not know how such intracomplex reactions depend on the link between enzyme and substrate. Here we show that the kinetics of tethered kinases follow a Michaelis-Menten-like dependence on effective concentration. We find that phosphorylation kinetics scale with the length of the intrinsically disordered linkers that join the enzyme and substrate but that the scaling differs between substrates. Steady-state kinetics can only partially predict rates of tethered reactions as product release may obscure the rate of phosphotransfer. Our results suggest that changes in signaling complex architecture not only enhance the rates of phosphorylation reactions but may also alter the relative substrate usage. This suggests a mechanism for how scaffolding proteins can allosterically modify the output from a signaling pathway.