The cAMP-dependent protein kinase (PKA) has been the most studied human protein kinase ever. Very recently, new X-ray crystallographic structures in which the SP20 substrate has been trapped in the ternary complex with PKA before and after the phosphoryl transfer have provided a few tentative snapshots of the evolution of the enzyme system along the catalytic reaction. In the present paper, we have studied the dissociative and the associative mechanisms for the phosphorylation reaction of the SP20 substrate catalyzed by PKA by means of MP2/aug-cc-pVTZ/CHARMM//B3LYP/6-31+G(d)/CHARMM electronic structure calculations using a complete solvated model of the PKAc-ATPMg 2 −SP20 system. Our results demonstrate that the dissociative mechanism (involving two consecutive steps: phosphoryl transfer and back protonation of the phosphorylated substrate) is clearly more favorable than the associative one. A comparison of Kemptide with SP20 shows that the catalytic mechanism is not substratedependent. However, the product complexes are better stabilized in the active site in the case of SP20, which may explain why phosphokemptide dissociates much faster. We show for the first time the viability of the SP20 phosphorylation process in a conformation of the PKAc-ATPMg 2 −SP20 ternary complex in which the Gly-rich loop is displaced with respect to the fully closed conformation of the PKAc-ATPMg 2 −IP20. Lastly, we provide a complete sequence of the geometrical evolution of the structure of the ternary complex along the catalytic reaction. This permits the identification of the snapshots corresponding to the above-mentioned new X-ray crystallographic structures, so validating the atomic view of the reaction suggested by them.
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.
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