SP20 Phosphorylation Reaction Catalyzed by Protein Kinase A: QM/MM Calculations Based on Recently Determined Crystallographic Structures

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-ATPMg₂-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 substrate-dependent. 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-ATPMg₂-SP20 ternary complex in which the Gly-rich loop is displaced with respect to the fully closed conformation of the PKAc-ATPMg₂-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. (Figure Presented).