An Adaptor Domain‐Mediated Autocatalytic Interfacial Kinase Reaction

Abstract

This paper describes a model system for studying the autocatalytic phosphorylation of an immobilized substrate by a kinase enzyme. This work uses self-assembled monolayers (SAMs) of alkanethiolates on gold to present the peptide substrate on a planar surface. Treatment of the monolayer with Abl kinase results in phosphorylation of the substrate. The phosphorylated peptide then serves as a ligand for the SH2 adaptor domain of the kinase and thereby directs the kinase activity to nearby peptide substrates. This directed reaction is intramolecular and proceeds with a faster rate than does the initial, intermolecular reaction, making this an autocatalytic process. The kinetic non-linearity gives rise to properties that have no counterpart in the corresponding homogeneous phase reaction: in one example, the rate for phosphorylation of a mixture of two peptides is faster than the sum of the rates for phosphorylation of each peptide when presented alone. This work highlights the use of an adaptor domain in modulating the activity of a kinase enzyme for an immobilized substrate and offers a new approach for studying biochemical reactions in spatially inhomogeneous settings.

A Spatially Propagating Biochemical Reaction

“…The experiment was repeated using a monolayer that presented the peptide at a density of 0.5 % and we measured a propagation velocity for the reaction of 0.51 μm min −1 (Figure 2 a). This lower velocity is consistent with our previous observation that the autocatalytic reaction has a strong dependence on substrate density, and shows a greater rate increase for monolayers that present the substrate at higher density 14. We have not determined whether sequential phosphorylation reactions are catalyzed by a single kinase—where the enzyme repeatedly dissociates and reassociates to the newly formed phosphopeptide—or whether new kinase enzymes are recruited from the bulk region.…”

ABL Tyrosine Kinases: Evolution of Function, Regulation, and Specificity

“…This result suggests a processive phosphorylation mechanism, that is to say, successive phosphorylation of different tyrosines on the same substrate (96, 97). Processivity would also explain why ABL-mediated phosphorylation shows accelerated, autocatalytic kinetics when substrate peptides are surface bound at high density (98). In this model, a newly phosphorylated tyrosine moves from the ABL catalytic site to the SH2 pocket, positioning another tyrosine residue for efficient phosphorylation.…”

Fluorescence Lifetime Imaging of Biosensor Peptide Phosphorylation in Single Live Cells

“…This effect likely arises from the more drastic change in the local environment of the fluorophore that could occur upon binding of the phosphopeptide with the protein, probably through the kinase SH2 domain. [14] …”