Quantum-Mechanical Study on the Catalytic Mechanism of Alkaline Phosphatases

Abstract

Alkaline phosphatases (APs) catalyze the hydrolysis and transphosphorylation of phosphate monoesters. The catalytic mechanism was examined by quantum-mechanical calculations using an active-site model based on the X-ray crystal structure of the human placental AP. Free energies of activation and of reaction for the catalytic steps were evaluated for a series of aryl and alkyl phosphate esters, and the computational results were compared with experimental values available in the literature. Mechanistic observations previously reported in experimental works were rationalized by the present theoretical study, particularly regarding the difference in the rate-determining step between aryl and alkyl phosphates. The formation rate of the covalent phosphoserine intermediate followed a linear free energy relationship (LFER) with the pK of the leaving group. This LFER, which could be experimentally determined only for less reactive alkyl phosphates, was verified by the present calculations to apply for the entire set of aryl and alkyl phosphate substrates.

Computational study on the role of residue Arg166 in alkaline phosphatases

“…The initial coordinates for the respective Michaelis complexes had been determined by previous molecular docking calculations. 10,12 In this complexes, the oxygen of the ester leaving group was coordinated to Zn 1 , and one nonbridging oxygen was coordinated to Zn 2 (Figure 1a). In the case of PLAP, the other two nonbridging oxygen atoms formed hydrogen bond interactions with the guanidinium group of Arg166 (the average O-H distances being 1.52 Å and 1.64 Å), and with a Mg 3 -bound water.…”

“…This reaction step has been determined as rate-limiting for the enzymatic hydrolysis of alkyl phosphates. [10][11][12][13] Calculations were performed for hydrogen, methyl, and ethyl phosphate dianions as substrates. Computed energy changes are shown in Table 1, and the structures of stationary points are illustrated in Figure 1.…”

“…22 This ONIOM(B3LYP:PM3MM) methodology has been verified to be suitable in our prior studies. [10][11][12] The electrostatic effect of the environment was taken into account by polarized continuum model (IEFPCM) 23 optimizations, considering a dielectric constant ε = 4.0 to simulate the influence of the residues surrounding the active site. The positions of backbone atoms involved in peptide bonds were fixed to conserve the active site structure, and the coordinates of the rest of the atoms were fully optimized.…”

“…9 Computational methods were recently applied to study the hydrolysis of phosphate esters within the active site of human placental AP (PLAP) as a model AP. [10][11][12] These calculations provided interesting insights regarding the catalytic mechanism of this family of enzymes. In this work, quantum mechanical calculations were performed with the aim of achieving a better understanding of the influence of Arg166 on the catalytic activity of APs.…”

Effect of Chemically Distinct Substrates on the Mechanism and Reactivity of a Highly Promiscuous Metallohydrolase

“…It has been suggested that hydrolysis of phosphomono-, phosphodi-, and phosphotriester substrates employ either the concerted D N A N or associative (A N + D N ) mechanism; a completely dissociative mechanism has not yet been observed for any of those substrate types. , The tightness of the transition state in these mechanisms, described in terms of the P–O­(nucleophile) and P–O­(leaving group) bond distances, decreases from mono- to triesters (Figure b) . Members of the binuclear metallohydrolase family have been proposed to employ a different mechanism depending on the nature of the substrate. ,,,− …”