SP20 Phosphorylation Reaction Catalyzed by Protein Kinase A : QM/MM Calculations Based on Recently Determined Crystallographic Structures
Pérez Gallegos, Ayax (Universitat Autònoma de Barcelona. Institut de Biotecnologia i de Biomedicina "Vicent Villar Palasí")
García Viloca, Mireia (Universitat Autònoma de Barcelona. Institut de Biotecnologia i de Biomedicina "Vicent Villar Palasí")
González Lafont, Àngels (Universitat Autònoma de Barcelona. Departament de Química)
Lluch, José M. (Universitat Autònoma de Barcelona. Departament de Química)

Data:	2015
Resum:	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 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-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.
Ajuts:	Ministerio de Ciencia e Innovación CTQ2011-24292 Ministerio de Economía y Competitividad CTQ2014-53144-P
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Llengua:	Anglès
Document:	Article ; recerca ; Versió acceptada per publicar
Matèria:	Associative mechanism ; Dissociative mechanism ; Enzyme catalysis ; General acid/base catalysis ; Phosphorylation reactions ; Protein kinase A ; QM/MM calculations
Publicat a:	ACS catalysis, Vol. 5, Num. 8 (August 2015) , p. 4897-4912, ISSN 2155-5435

DOI: 10.1021/acscatal.5b01064

41 p, 1.6 MB

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