Enzymatic activity of alkaline phosphatases: a quantum-chemical study.

BOROSKY, GABRIELA L.

Montevideo

Congreso; XLII Congreso de Químicos Teóricos de Expresión Latina, Quitel 2016; 2016

Alkaline phosphatases (APs) are metalloenzymes found in many species from bacteria to man, which catalyze the hydrolysis of phosphomonoesters with release of inorganic phosphate and an alcohol. The catalytic mechanism involves activation of a serine residue, formation of a covalent phosphoseryl intermediate, hydrolysis of the phosphoserine by an activated water molecule, and release of the phosphate product or its transfer to a phosphate acceptor.In this work, computational calculations were performed with the aim of achieving a better understanding of the catalytic activity of APs. Human placental AP (PLAP), one of the four AP isozymes found in human, was used as a model protein. The active site of the enzyme was built according to the crystal structure reported in the Protein Data Bank. The reaction steps of the catalytic mechanism were evaluated within the active site for several phosphomonoesters substrates, analyzing the effect of the type of substrate (aromatic or aliphatic) on the catalytic activity. Quantum-mechanical methods were applied by using a two-layer ONIOM(QM:QM) procedure. In this way, density functional theory (DFT) computations were carried out for the reacting system (phosphomonoester substrate and catalytic serine), as well as for the metal cations, their ligands, and the water molecules involved in the catalytic mechanism, while semiempirical methods were employed for the rest of the atoms. The influence of the protein environment was taken into account by means of the IEFPCM method. Free energies of reaction (ΔGr) and of activation (ΔG≠) 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.