Abstract

Mg2+ ions are essential for the proper functioning of protein kinases, and their roles in kinase activity have been studied for years. However, recent investigations have shed light on how these metal cofactors modulate the catalytic activity, and other functions for them have been assigned. As an example, it has been found that in cyclin-dependent kinase 2 (CDK2), an enzyme that had been postulated to work efficiently with only one Mg2+ ion, a second Mg2+ ion needs to be bound in the active site for achieving optimal catalytic performance. In this contribution, the phosphoryl transfer reaction in CDK2 has been studied in detail considering the presence of an additional Mg2+ ion in the active site. For this purpose, quantum mechanics/molecular mechanics (QM/MM) free energy calculations with the adaptive string method were performed, which showed that indeed the system containing two Mg2+ ions exhibits a lower activation free energy, corroborating the experimental observations. Structural and electronic analyses helped identify the main factors that explain the differences in reaction barriers, emphasizing the reduced electrostatic repulsion that is felt by the reacting fragments when two Mg2+ ions are present in the active site. On the other hand, it was confirmed that the base-assisted mechanism is favored over the substrate-assisted pathway in the presence of two Mg2+ ions. The role of Asp127 was clarified: this residue acts first as a catalytic base and then as a catalytic acid protonating the transferred phosphoryl group. It is expected that these results may be extrapolated to other structurally related kinases where the influence of a second Mg2+ ion within the active site is still under debate.