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.

Figure 1

(A) Model system for the auto-catalytic phosphorylation of an immobilized peptide substrate by a kinase that includes an adaptor domain that recognizes its phosphopeptide product. The kinase can phosphorylate the peptide substrate by way of an ‘intermolecular’ pathway (with rate constant k₁) or an ‘intramolecular’ pathway (with rate constant k₂). (B) SAMDI mass spectrometry was used to characterize the phosphorylation of a peptide immobilized to a monolayer by Abl kinase. A spectrum of the monolayer prior to reaction showed a peak at m/z of 2203, corresponding to the peptide-terminated disulfide. Following treatment with Abl kinase, a mass spectrum of themonolayer revealed a new peak at m/z of 2283, resulting from phosphorylation of the peptide.

Figure 2

(A) A time course of the reaction shown in Figure 1 revealed that the phosphorylation reaction was initially slow and then accelerated after accumulation of a minor amount of product (◆). A corresponding time course for a reaction on a monolayer that presents a mixture of peptide and phosphopeptide showed a much shorter lag phase (▲). The data were fit with equation 1 as described in the text. (B) The reaction was repeated in the presence of a soluble inhibitor of the SH2 adaptor domain. The interfacial reaction showed a dose-dependent inhibition in rate (◆) while an analogous reaction performed in solution was unaffected by the inhibitor (▲). The data for the interfacial reaction were fit to a sigmoidal curve described by y=min + (max-min)EC50/(EC50 + 10^x) with the minimum (min) and maximum (max) values of y and the concentration of ligand at half-maximum response (EC50) as adjustable parameters. The data for the homogeneous phase reaction were fit to a straight line.

Figure 3

The kinase reaction was performed in the presence of a soluble phosphopeptide (I) for concentrations ranging from 0 to 1 mM. The solid curves are simulations derived from equation (1) with k₁=0.0079 min^-1, k₂=0.525 min^-1 and K_d=40 μM.

Figure 4

(A) Phosphorylation of two peptide substrates in solution follows Michaelis-Menten kinetics. Peptide I (◆) is a more active substrate for Abl kinase than is peptide II (▲). (B) The two peptides were separately immobilized to monolayers and treated with Abl. Kinetic profiles for the reactions were fit with equation 1 and gave values of k₁ and k₂.

Figure 5

A comparison of the rates for phosphorylation of substrate II when present alone (◆) or as a mixture with substrate I (▲). The phosphorylation of substrate II displays a shorter lag phase and a greater maximum rate when it is present as a mixture with substrate I. For the mixed monolayer, the rate for phosphorylation of substrate I is also shown (●).

Figure 6

(A) A series of monolayers presenting peptide substrate at densities of 1, 2.5, 5 and 10% were treated with kinase for times ranging from 0 to 60 minutes. The kinetic profiles depend on the density of the substrate and show little auto-amplification at low density. (B) A model to approximate the number of peptide substrates that are available to a kinase that is tethered to the monolayer.