Resumen de Dynamics, predicted and not, of a system of biomedical interest: an insight into the pka
Protein Kinases are highly widespread in nature. There are more than 500 protein kinase genes identified representing, more or less, 1.7% of all human genes. They catalyze the protein phosphorylation, a reversible covalent modification that regulates a large number of aspects of cell life: cellular regulation, metabolic pathways, gene transcription, signal transduction,etc.
The protein kinase family is large and structurally diverse, but all the enzymes of this group have a highly conserved catalytic core with a bilobal structure that suggests the same catalytic mechanism. For all this reasons, we selected the Protein Kinase A (PKA) as the object of our study, which is the most experimentally studied enzyme of the protein kinase family.
Two options have been suggested for the phosphoryl transfer mechanism and up to now it is not clear which of them is the actual one: the first pathway consists in a concerted phosphoryl and proton-transfer process between ATP (the cofactor) and the substrate (always a specific serine, threonine or tyrosine residue); the second one is a dissociative path in which the substrate proton is first accepted by the enzyme's aspartate 166 (Asp166), and then the phosphoryl group passes from ATP to the substrate. Different research groups suggest the second pathway as the most reliable one.
We have tested two substrates in this study: the first is the synthetic heptameric peptide, LRRASLG (kemptide), which corresponds to the consensus sequence with a serine at the fifth position (Ser17); the second one is the 20-residue peptide SP20. Both have been modeled in the active center of the PKA enzyme by manually modifying the PKI inhibitor, which is bound to the enzyme in most X-ray structures and in the two structures we selected to build the models.
Additionally, we have considered different phosphorylation states of the enzyme (one phosphorylation site modified, two phosphorylation sites modified, or no phosphorylation at all) and a mutant of PKA to test the effect of small modification on the activity of the system.
The thermodynamics and kinetics of the PKA-ATP-kemptide complex has been experimentally determined, thus it is, in our opinion, the perfect model to theoretically study which of the pathways is the most favorable.
Finally, after an analysis of the behaviour along Molecular Dynamics simulations of the five models we have built, we have selected two of them, thus excluding the SP20 substrate, the mutant, and the deactivated form, to study the potential energy surfaces corresponding to both mechanisms. We have used different techniques and different levels of theory (a modified AM1/d semiempirical method and a DFT method) to achieve optimum results. The semiempirical method choice, followed an accurate analysis of its performance on a rather large set of molecules and model reactions, meant to simulate the phosphorylation reaction catalyzed by the PKA.
