Dual Role of Magnesium Ions in Active pCDK2/Cyclin: Essential Activator of Kinase Activity and Partial Inhibitor of Product Release

Abstract

Protein kinases play a critical role in the signaling pathways the regulation of nearly all biological processes. The activity of protein kinases is strictly regulated by a number of factors and one of these is the availability of divalent metals which are essential to the reaction. The activity of many protein kinases has been shown to be sensitive to [Mg2+], with a variety of different kinetic mechanisms proposed to explain this dependence. In this work we dissect the multiple roles that the two essential Magnesium ions play in the catalytic mechanism of CDK2. In combination with our previous work on the transition state mimic of CDK2, we now present a crystal structure of ADP bound to pCDK2/Cyclin featuring two Mg2+ ions, molecular dynamics calculations at several stages of the catalytic cycle, and extensive steady state kinetics to highlight different features of the kinetic mechanism as a function of [Mg2+]. We find that the binding of the second Mg2+ to the active site is essential to the progression of catalysis, and that this metal promotes stability of the ATP∗Mg substrate as well as the transition state of the reaction. At the same time, the occupancy of the second Mg2+ site also stabilizes the ADP∗Mg product following catalysis and thus also partially inhibits further rounds of catalysis. We have constructed a mathematical model of the reaction kinetics as a function of [ATP∗Mg] and [Mg]. We propose that the form of this model may be of general application to protein kinases, and that the varied responses of different protein kinases to [Mg2+] can be represented in by altered values of the model parameters.