A Transition Path Ensemble Study Reveals a Linchpin Role for Mg2 during Rate-Limiting ADP Release from Protein Kinase A

作者：Khavrutskii Ilja V^*; Grant Barry; Taylor Susan S; McCammon J Andrew

来源：Biochemistry, 2009, 48(48): 11532-11545.

DOI：10.1021/bi901475g

Protein kinases are key regulators of diverse signaling networks critical for growth and development. Protein kinase A (PKA) is all important kinase prototype that phosphorylates protein targets at Ser and Thr residues by converting ATP to ADP. Mg2 ions play a crucial role in regulating phosphoryl transfer and can limit overall enzyme turnover by affecting ADP release. However, the mechanism by which Mg2 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 Mg2 -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 Mg2 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 Mg2 ions restricts mobility of the Gly-rich loop that closes over the active site. We find that simultaneous release of ADP with Mg2 ions from the active site is unfeasible. Thus, we conclude that Mg2 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 Mg2 -dependent association of nucleotides with protein kinases.

• 出版日期2009-12-8