title: 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.
authors.affiliation: Dipartimento di Chimica, Università della Calabria,Via P. Bucci, cubo 14c, 87036 Arcavacata di Rende (CS), Centro di Calcolo ad Alte Prestazioni per Elaborazioni Parallele e Distribuite-Centro d'Eccellenza MIUR, Italy and REQUIMTE, Departamento de Química, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, 687, 4169-007 Porto, Portugal.
authors.forename: Marta E
authors.initials: ME
authors.lastname: Alberto
authors.affiliation: Dipartimento di Chimica, Università della Calabria,Via P. Bucci, cubo 14c, 87036 Arcavacata di Rende (CS), Centro di Calcolo ad Alte Prestazioni per Elaborazioni Parallele e Distribuite-Centro d'Eccellenza MIUR, Italy and REQUIMTE, Departamento de Química, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, 687, 4169-007 Porto, Portugal.
authors.forename: Tiziana
authors.initials: T
authors.lastname: Marino
authors.affiliation: Dipartimento di Chimica, Università della Calabria,Via P. Bucci, cubo 14c, 87036 Arcavacata di Rende (CS), Centro di Calcolo ad Alte Prestazioni per Elaborazioni Parallele e Distribuite-Centro d'Eccellenza MIUR, Italy and REQUIMTE, Departamento de Química, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, 687, 4169-007 Porto, Portugal.
authors.forename: Maria J
authors.initials: MJ
authors.lastname: Ramos
authors.affiliation: Dipartimento di Chimica, Università della Calabria,Via P. Bucci, cubo 14c, 87036 Arcavacata di Rende (CS), Centro di Calcolo ad Alte Prestazioni per Elaborazioni Parallele e Distribuite-Centro d'Eccellenza MIUR, Italy and REQUIMTE, Departamento de Química, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, 687, 4169-007 Porto, Portugal.
authors.forename: Nino
authors.initials: N
authors.lastname: Russo