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Recent experimental results have challenged conventional views on the role metals play in the chemistry of protein kinases because it has been shown that (cAMP)-dependent protein kinase (PKA) is active in the presence of other divalent alkaline earth metal cations besides physiological Mg 2+ ions. This has raised the important possibility that Ca 2+ may also be a physiological cofactor of protein kinases. In this work, QM/MM calculations, at the DFT and MP2 levels for the QM part, on complete solvated models of PKAc-M 2 ATP-substrate ternary complexes, with PKAc as the catalytic subunit of PKA, M denoting Ca 2+ or Sr 2+ and substrate denoting SP20 or Kemptide, have been carried out for the overall phosphoryl transfer reaction. In accordance with the experimental data, our theoretical results show for the first time at the molecular level how the overall PKAc-catalyzed phosphorylation of SP20, via a dissociative mechanism, is plausible with Ca 2+ and Sr 2+ . The viability of the catalytic reaction with Kemptide and Ca 2+ is also verified here. The energy barrier of the rate-limiting phosphoryl-transfer step does not depend on different coordination environments of the alkaline earth metal cations whereas the proton-transfer step region is metal dependent making the global chemical process more exoergic on going from Mg 2+ to Sr 2+ . This trend is in agreement with the less effective release of the phosphorylated product observed experimentally in the presence of Ca 2+ versus Mg 2+ , and would explain also the lower activity of PKAc with Ca 2+ , since phospho-substrate and ADP releases are rate limiting for catalytic turnover. For the same reason, we predict an even lower activity of PKAc with Sr 2+ . Moreover, the active sites of the in silico reactant and product complexes and the available X-ray crystallographic structures show good agreement.

In this work a theoretical study of the γ-phosphoryl group transfer from ATP to Ser17 of the synthetic substrate Kemptide (LRRASLG) in protein kinase A (PKA) has been carried out with a solvated model of the PKA-Mg2ATP-Kemptide system based on the X-ray crystallographic structure. We have used high levels (B3LYP/MM and MP2/MM) of theory to determine the overall reaction paths of the so-called concerted loose mechanism trying to clarify some aspects of that mechanism still under debate. Our calculations demonstrate for the first time in a complete model of the ternary system the viability of the final step of the catalytic mechanism in which the protonation of the phosphokemptide product by Asp166 takes place. Asp166 is a base catalyst that abstracts the HγSer17 of Kemptide thus facilitating the phosphoryl transfer, but it also acts as an acid catalyst by donating the proton just accepted from Ser17 to the O2γATP atom of the phosphoryl group.

Here we analyze in detail the possible catalytic role of the associative mechanism in the γ-phosphoryl transfer reaction in the catalytic subunit of the mammalian cyclic AMP-dependent protein kinase (PKA) enzyme and its D166A mutant. We have used a complete solvated model of the ATP-Mg2-Kemptide/PKA system and good levels of theory (B3LYP/MM and MP2/MM) to determine several potential energy paths from different MD snapshots, and we present a deep analysis of the interaction distances and energies between ligands, metals and enzyme residues. We have also tested the electrostatic stabilization of the transition state structures localized herein with the charge balance hypothesis. Overall, the results obtained in this work reopen the discussion about the plausibility of the associative reaction pathway and highlight the proposed role of the catalytic triad Asp166-Lys168-Thr201.

cAMP-dependent protein kinase (PKA) is one of the simplest and best understood members of the protein kinase family. In a previous study, we have theoretically studied the complex between PKA and the heptapeptide substrate Kemptide by classical molecular dynamics. On the basis of the results obtained for Kemptide, the aim of the present work is to explore how the different conditions, such as phosphorylation state, substrate, and mutations of key residues affect the enzyme dynamics. We have built different models of the complex; particularly we have focused our attention on two crystallographic structures which main difference consists in their phosphorylation state. The first one has the residue Thr197 modified into a phospho-threonine (pThr197); the second one, in addition to the same Thr197, has also the residue Ser338 modified into a phospho-serine (pSer338). In addition, we have analyzed the effect of the choice of the substrate by building a model of the PKA-SP20 Michaelis complex. Finally, we have theoretically studied the effect of the mutation of the highly conserved residue Asp166 that, experimentally, leads to a decrease of the reaction rate. The results of this study give insight into the dynamical states of the enzyme and their relationship with different elements of the model, which correspond to different natural or human guided situations of the active biological system., (Copyright © 2011 Elsevier B.V. All rights reserved.)

We present here a theoretical study of the phosphoryl transfer catalytic mechanism of protein kinase A, which is the best known member of the large protein kinase family. We have built different theoretical models of the complete PKA-Mg(2)-ATP-substrate system to explore the two most accepted reaction pathways, using for the first time in a reaction mechanism theoretical study, the heptapeptide substrate Kemptide, which is relevant for its high efficiency and small size. The effect of the protein configuration, as modeled by two different X-ray structures with different phosphorylation states and degrees of flexibility, has been analyzed. The results indicate that the environmental conditions can influence the availability of the pathways and thus the choice of the mechanism to be followed. In addition, the roles of the two active site conserved residues, Asp166 and Lys168, have been analyzed for each reaction mechanism.

In the present work we have modeled the Michaelis complex of the cyclic-Adenosine Monophosphate Dependent (cAMD) Protein Kinase A (PKA) with Mg(2)ATP and the heptapeptide substrate Kemptide by classical molecular dynamics. The chosen synthetic substrate is relevant for its high efficiency and small size, and it has not been used in previous theoretical studies. The structural analysis of the data generated along the 6 ns simulation indicates that the modeled substrate-enzyme complex mimics the substrate binding pattern known for PKA. The values of the average prereactive distances obtained from the simulation do not exclude any of the two limiting situations proposed as mechanisms in the literature for the phosphorylation reaction (dissociative and associative) because the system oscillates between configurations compatible with each of them. Furthermore, the results obtained for the average interaction distances between active site residues concord in suggesting the plausibility of an alternative third reaction mechanism.