Atomistic details of the Catalytic Mechanism of Fe(III)−Zn(II) Purple Acid Phosphatase

Abstract

In the present work, we performed a theoretical investigation of the reaction mechanism of the Fe(III)-Zn(II) purple acid phosphatase from red kidney beans (rkbPAP), using the hybrid density functional theory and employing different exchange-correlation potentials. Characterization of the transition states and intermediates involved and the potential energy profiles for the reaction in different environments (gas phase, protein environment, and water) are reported. Our results show that the Fe(III)-Zn(II)PAP catalyzes the hydrolysis of methylphosphate via direct attack by a bridging metals-coordinated hydroxide leading to the cleavage of the ester bond. From our study emerges that the rate-limiting step of the reaction is the nucleophilic attack followed by the less energetically demanding release of the leaving group. Furthermore, we provide insights into some important points of contention concerning the precatalytic complex and the substrate coordination mode into the active site prior to hydrolysis. In particular: (i) Two models of enzyme-substrate with different orientations of the substrate into the active site were tested to evaluate the possible roles played by the conserved histidine residues (His 202 and His 296); (ii) Different protonation states of the substrate were taken into account in order to reproduce different pH values and to verify its influence on the catalytic efficiency and on the substrate binding mode; (iii) The metals role in each step of the catalytic mechanism was elucidated. We were also able to ascertain that the activation of the leaving group by the protonated His 296 is decisive to reach an optimal catalytic efficiency, while the bond scission without activation requires higher energy to occur.

The Catalytic Mechanism of Protein Phosphatase 5 Established by DFT Calculations

“…The activation energy for this step, calculated at the IEFPCM MPWB1K/6‐311+G(d,p)|SDD level in the antiferromagnetic coupling is 15.8 kcal mol −1 (see Figure 3 and Table 2 in the Supporting Information). This energy is consistent with the experimental data4b and similar studies on related enzymes 19. Actually, the available experimental data regarding the catalytic mechanism of PP5 is not complete.…”

“…The 6‐31G(d,p)24 basis set has been chosen for the C, N, O, P and H atoms, while for the metal the SDD25 pseudopotential has been used. This combination of functional and basis sets was used before with success in similar mechanistic studies with metal cofactors 19. 26…”

“…MPWB1K was chosen according to previous DFT benchmarking studies on similar systems, in which we showed that MPWB1K generates accurate activation energies for the cleavage of phosphoester bond reactions 19. 26a, 29 Implicit solvation was included with the IEFPCM continuum model 30.…”

How Can Methanol Dehydrogenase from Methylacidiphilum fumariolicum Work with the Alien CeIII Ion in the Active Center? A Theoretical Study

“…The solvation energies were obtained from single point calculations performed on the optimized geometries with the more extended basis set 6–311+G(2d,2p)|SDD. The choice of the dielectric constant ϵ =4 is usually considered to be a good representation of protein surrounding . For the potential energy surfaces (PESs), the final solvation energies reported include the ZPE corrections.…”

chem‐path‐tracker: An Automated Tool to Analyze Chemical Motifs in Molecular Structures

“…Generally, only a small set of residues of the active site is analyzed either because those are the ones that are directly involved in the catalytic process or because they have somehow been shown to be important for the reaction. The surrounding residues are most of the time disregarded, although several studies have shown that these residues are equally important for the function of the enzyme, as they maintain the reactive residues close to each other and they can also change the chemistry of neighbor residues .…”