The robustness of proofreading to crowding-induced pseudo-processivity in the MAPK pathway

TL;DR

This paper investigates how crowding-induced pseudo-processivity affects the specificity and proofreading ability of double phosphorylation in the MAPK pathway, revealing that increased rebinding reduces specificity but does not impair proofreading.

Contribution

It demonstrates that pseudo-processivity due to rebinding decreases substrate specificity but does not compromise the proofreading function of double phosphorylation.

Findings

Increased rebinding reduces substrate specificity.

Pseudo-processivity does not impair proofreading effectiveness.

Loss of specificity is due to intrinsic selectivity reduction, not pseudo-processivity.

Abstract

Double phosphorylation of protein kinases is a common feature of signalling cascades. This motif may reduce cross-talk between signalling pathways, as the second phosphorylation site allows for proofreading, especially when phosphorylation is distributive rather than processive. Recent studies suggest that phosphorylation can be `pseudo-processive' in the crowded cellular environment, as rebinding after the first phosphorylation is enhanced by slow diffusion. Here, we use a simple model with unsaturated reactants to show that specificity for one substrate over another drops as rebinding increases and pseudo-processive behavior becomes possible. However, this loss of specificity with increased rebinding is typically also observed if two distinct enzyme species are required for phosphorylation, i.e. when the system is necessarily distributive. Thus the loss of specificity is due to an intrinsic reduction in selectivity with increased rebinding, which benefits inefficient reactions, rather than pseudo-processivity itself. We also show that proofreading can remain effective when the intended signalling pathway exhibits high levels of rebinding-induced pseudo-processivity, unlike other proposed advantages of the dual phosphorylation motif.