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

BOROSKY, GABRIELA L.

Rio de Janeiro

Encuentro; Theoretical Chemistry in Rio; 2012

Alkaline phosphatases (APs) are metalloenzymes found in many species from bacteria to man. They catalyse the hydrolysis of phosphomonoesters with release of inorganic phosphate and alcohol. The catalysis involves the activation of the catalytic serine, the formation of a covalent phosphoseryl intermediate, the hydrolysis of the phosphoseryl by an activated water molecule, and the 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. In this work computational methods were applied with the aim of achieving a better understanding of the catalytic mechanism of PLAP. 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 environment. Quantum-chemical calculations were performed employing the ONIOM procedure. In this way, DFT computations were carried out for the reacting phosphomonoester and catalytic serine, as well as for the metal cations, their ligands, and the water molecules involved in the catalytic mechanism, while a semiempirical hamiltonian was applied for the rest of the atoms. Free energies of activation (?¢G??) and of reaction (?¢Gr) were evaluated for each step of the mechanism of catalysis. Mechanistic variations according to the nature of the substrate (an aryl or alkyl phosphate) were examined.