Robust regulation of protein kinases (PK) is critical for their central role in signal transduction pathways. Many oncogenes harbor mutations that misregulate PK activity. The chemical basis for how some PK regulatory factors ultimately affect the rate of chemistry is still not completely understood. We have identified a long-range electrostatic switch that we believe is used by allosteric PK regulatory factors to modulate the rate of chemistry by tuning active-site dynamics.We applied a combination of molecular dynamics simulation, crystallography, and kinetics to determine the chemical basis for how this electrostatic switch, toggled by regulatory subunit binding, affects each step of CDK2 catalysis. We engineered point mutants to deconstruct the kinetic, dynamic, and thermodynamic consequences of the switch. We also evaluated other PKs and find that, although it has evolved to be triggered by diverse PK regulatory factors, the switch mechanics are conserved.A key component of the switch is that it affects a significant change in the electrostatic potential within the ATP*Mg binding site. This electrostatic effect is propagated through the low-dielectric protein interior and directly affects the two dominant rate-determining steps of catalysis: attenuating both the recruitment of catalytically essential Mg cofactors (affecting both kcat and KM) as well as release of ADP.Conclusion: We present a chemical hypothesis that explains how a large-scale conformation transition, observed in diverse PK family members, mechanistically controls catalytic rate via electrostatics.

Allosteric regulation of protein kinase enzymes via an electrostatic switch that modulates active site dynamics

248th National Meeting (2014)

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