Alkaline phosphatases: a quantum-mechanical study on the influence of some active site residues.

BOROSKY, GABRIELA L.

Congreso; XLIV Congreso Internacional de Químicos Teóricos de Expresión Latina, Quitel 2018; 2018

Alkaline phosphatases (APs) constitute a family of metalloproteins found in numerous organisms from bacteria to man [1]. These enzymes catalyze the hydrolysis of phosphomonoesters, releasing inorganic phosphate and an alcohol [2]. The catalytic mechanism involves activation of a serine amino acid, formation of a covalent phosphoseryl intermediate, hydrolysis of the phosphoserine by an activated water molecule, and release or transfer of a phosphate dianion.Computational calculations were performed with the goal of achieving a better understanding of the catalytic influence of some significant residues of the active site of APs. Human placental AP (PLAP), one of the four human AP isozymes, 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 aromatic and aliphatic phosphomonoester substrates. 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) and more relevant atoms, while semiempirical methods were employed for the rest of the system. The influence of the protein environment was considered by means of the IEFPCM continuum method. Thermodynamic and kinetic parameters were computed for each step of the mechanism of catalysis, and the effect of replacing some keynote amino acids by other residues (mutations) was analyzed.