In silico study on the catalytic mechanism of human placental alkaline phosphatase.

BOROSKY, GABRIELA L.

Santiago

Congreso; 10th Congress of the World Association of Theoretical and Computational Chemists, WATOC 2014; 2014

Alkaline phosphatases (APs) are metalloenzymes found in many species from bacteria to man.[1] They catalyze the hydrolysis of phosphomonoesters with release of inorganic phosphate and alcohol.[2] The catalysis involves activation of the catalytic serine, formation of a covalent phosphoseryl intermediate, hydrolysis of the phosphoseryl by an activated water molecule, and release of the phosphate product or its transfer to a phosphate acceptor. Human placental AP (PLAP) is one of the four AP isozymes found in human. The aim of this work was to achieve a better understanding of the catalytic mechanism of PLAP by means of computational methods. The active site of PLAP was modelled according to the crystal structure reported in the Protein Data Bank. The reaction steps of the catalytic mechanism were evaluated within the active site. Quantum-chemical calculations were performed employing a two-layer QM/QM-ONIOM procedure. In this way, density functional theory (DFT) computations were carried out for the reacting system (phosphomonoester and catalytic serine), as well as for the metal cations, their ligands, and the water molecules involved in the catalytic mechanism. On the other hand, semiempirical methods were employed for the rest of the atoms. Different density functionals and semiempirical methods were applied and compared. Free energies of activation (ΔG≠) and of reaction (ΔGr) were computed for each step of the mechanism of catalysis. Mechanistic variations according to the nature of the substrate (an aryl or alkyl phosphate) were examined. The influence of the protein environment was considered with the IEFPCM method. Acknowledgments: G. L. Borosky acknowledges CONICET and Secyt-UNC for financial support. References: [1] R. B. McComb, G. N. Bowers, S. Posen, in Alkaline Phosphatases, Plenum Press, New York, (1979) 986 [2] J. H. Schwartz, F. Lipmann, Proc. Natl. Acad. Sci. USA 47 (1961) 1996.