A transition path ensemble study reveals a linchpin role for Mg(2+) during rate-limiting ADP release from protein kinase A

Biochemistry. 2009 Dec 8;48(48):11532-45. doi: 10.1021/bi901475g.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Adenosine Diphosphate / chemistry
  • Adenosine Diphosphate / metabolism*
  • Adenosine Triphosphate / chemistry
  • Adenosine Triphosphate / metabolism
  • Algorithms
  • Binding Sites
  • Cations, Divalent
  • Cyclic AMP-Dependent Protein Kinases / chemistry
  • Cyclic AMP-Dependent Protein Kinases / metabolism*
  • Enzyme Activation
  • Glycine / chemistry
  • Glycine / metabolism
  • Kinetics
  • Magnesium / chemistry
  • Magnesium / metabolism*
  • Models, Molecular
  • Serine / chemistry
  • Serine / metabolism
  • Thermodynamics
  • Threonine / chemistry
  • Threonine / metabolism

Substances

  • Cations, Divalent
  • Threonine
  • Serine
  • Adenosine Diphosphate
  • Adenosine Triphosphate
  • Cyclic AMP-Dependent Protein Kinases
  • Magnesium
  • Glycine