Protein kinases are key regulators of diverse signaling networks critical for growth and development. Protein kinase A (PKA) is an important kinase prototype that phosphorylates protein targets at Ser and Thr residues by converting ATP to ADP. Mg(2+) ions play a crucial role in regulating phosphoryl transfer and can limit overall enzyme turnover by affecting ADP release. However, the mechanism by which Mg(2+) participates in ADP release is poorly understood. Here we use a novel transition path ensemble technique, the harmonic Fourier beads method, to explore the atomic and energetic details of the Mg(2+)-dependent ADP binding and release. Our studies demonstrate that adenine-driven ADP binding to PKA creates three ion-binding sites at the ADP/PKA interface that are absent otherwise. Two of these sites bind the previously characterized Mg(2+) ions, whereas the third site binds a monovalent cation with high affinity. This third site can bind the P-3 residue of substrate proteins and may serve as a reporter of the active site occupation. Binding of Mg(2+) ions restricts mobility of the Gly-rich loop that closes over the active site. We find that simultaneous release of ADP with Mg(2+) ions from the active site is unfeasible. Thus, we conclude that Mg(2+) ions act as a linchpin and that at least one ion must be removed prior to pyrophosphate-driven ADP release. The results of the present study enhance understanding of Mg(2+)-dependent association of nucleotides with protein kinases.