A Michaelis-Menten Equation for Intra-Complex Kinase Reactions

Abstract

Kinase specificity is crucial to the fidelity of signalling pathways, but many kinases are surprisingly promiscuous and take part in widely different pathways. The intrinsic specificity of the enzymatic domain is augmented by a myriad of anchoring proteins that physically tether the kinase to certain substrates. Tethered enzyme reactions are independent of substrate concentration, and tethering can increase catalytic rates by orders of magnitude. Quantitative models for enzyme kinetics such as e.g. the Michaelis-Menten equation describe catalytic rates as a function of the substrate concentration, and are thus inherently unable to describe concentration-independent reactions. Here we develop a quantitative model for phosphorylation rates by a tethered kinase. We study a model system consisting of the catalytic subunit of protein kinase A tethered to its substrate by intrinsically disordered linkers. Using single-turnover kinetics, we show that 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 only partially predict rates of tethered reactions as product release may obscure the rate of phospho-transfer. Our results suggest that changes in signalling complex architecture not only enhance the rates of phosphorylation reactions, but may also alter the relative substrate usage. This suggests a mechanism for how anchoring proteins can allosterically modify the output from a signalling pathway.