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

Abstract

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.

Metal Fluorides as Analogues for Studies on Phosphoryl Transfer Enzymes

“…The resulting structures have then permitted computations aimed at delineating the molecular mechanisms of a variety of enzymes catalyzing PTx reactions, [40] particularly the small GTPases, which play critical roles in cell signaling and regulation, and to cAPK. [41] Theoretical methods provide considerable insights into the distribution of electrons within molecules, and the energies of protein/ligand interactions that mediate binding and TS stabilization. [42] Calculations have also been used to obtain accurate structures that were used to resolve the nature of MFx species in x-ray crystal structures of relatively low resolution.…”

Theoretical Study of the Phosphoryl Transfer Reaction from ATP to Dha Catalyzed by DhaK from Escherichia coli

“…23,24 Studies of the phosphate transfer from ATP to serine or tyrosine residues in protein kinases have been also widely reported. 3,[25][26][27][28][29][30][31] Thus, a theoretical study in cyclindependent kinase (CDK2) by De Vivo et al 25 proposed that an Asp residue located in the active site would play a structural role whereas the activation of the Ser nucleophile was proposed to be due to a proton transfer to ATP. The authors suggested a substrateassisted mechanism through a single concerted transition state (TS).…”

Theoretical Studies of the Catalytic Mechanism of the Dihydroxyacetone Kinase

“…There is an extensive debate which still remains open about the most favorable phosphoryl transfer mechanism in kinase enzymes. Some authors consider the asp-assisted mechanism as the most favorable one 48,52,[87][88][89][90] while others support the substrate-assisted mechanism as the most likely.…”

“…There are several computational studies in cAMP-dependent protein kinase (or PKA), an enzyme that catalyse the γ-phosphoryl transfer from ATP to a Ser or Thr residue of a substrate peptide. 48,[87][88][89] For example, Diaz and Field 87 conducted classical MD simulations of the catalytic subunit of PKA, Mg2ATP and a 20-residue peptide substrate, using the CHARMM force field. They showed that the interaction between the carboxylate group of Asp residue and the nucleophilic Ser was dynamically stable.…”

“…In fact, the latter was suggested to be the role for the Lys residue, keeping the ATP and the substrate peptide in a proper conformation for the nucleophilic attack. Another examples where the asp-assisted mechanism has been analyzed in PKA are the more recently theoretical works of Pérez-Gallegos et al 48,89 They explored potential energy profiles of the phosphoryl transfer mechanism using QM/MM hybrid methods with DFT (B3LYP/6-31+G(d)) and MP2 functionals. Authors studied the phosphoryl transfer in two different substrates; the heptapeptide synthetic substrate, Kemptide 48 and the 20-residue, SP20 89 substrate.…”