title: Proton shuttles and phosphatase activity in soluble epoxide hydrolase.
abstract: Recently, a novel metal Mg2+-dependent phosphatase activity has been discovered in the N-terminal domain of the soluble epoxide hydrolase (sEH), opening a new branch of fatty acid metabolism and providing an additional site for drug targeting. Importantly, the sEH N-terminal fold belongs to the haloacid dehalogenase (HAD) superfamily, which comprises a vast majority of phosphotransferases. Herein, we present the results of a computational study of the sEH phosphatase activity, which includes classical molecular dynamics (MD) simulations and mixed quantum mechanical/molecular mechanics (QM/MM) calculations. On the basis of experimental results, a two-step mechanism has been proposed and herein investigated: (1) phosphoenzyme intermediate formation and (2) phosphoenzyme intermediate hydrolysis. Building on our earlier work, we now provide a detailed description of the reaction mechanism for the whole catalytic cycle along with its free energy profile. The present computations suggest metaphosphate-like transition states for these phosphoryl transfers. They also reveal that the enzyme promotes water deprotonation and facilitates shuttling of protons via a metal-ligand connecting water bridge (WB). These WB-mediated proton shuttles are crucial for the activation of the solvent nucleophile and for the stabilization of the leaving group. Moreover, due to the conservation of structural features in the N-terminal catalytic site of sEH and other members of the HAD superfamily, we suggest a generalization of our findings to these other metal-dependent phosphatases.
authors.affiliation: Department of Chemistry, University of Pennsylvania, 231 South 34th Street, Philadelphia, Pennsylvania 19104-6323, USA. mdevivo@cmm.upenn.edu
authors.forename: Marco
authors.initials: M
authors.lastname: De Vivo
authors.forename: Bernd
authors.initials: B
authors.lastname: Ensing
authors.forename: Matteo
authors.initials: M
authors.lastname: Dal Peraro
authors.forename: German A
authors.initials: GA
authors.lastname: Gomez
authors.forename: David W
authors.initials: DW
authors.lastname: Christianson
authors.forename: Michael L
authors.initials: ML
authors.lastname: Klein
mesh_terms: D002384:Catalysis; D003198:Computer Simulation; D004789:Enzyme Activation; D004851:Epoxide Hydrolases / Q000737:chemistry*; D006868:Hydrolysis; D008958:Models, Molecular; D008968:Molecular Conformation; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry*; D017433:Protein Structure, Secondary; D011522:Protons; D011789:Quantum Theory; D012995:Solubility
chemical_list: D011522:Protons; D010744:Phosphoric Monoester Hydrolases; D004851:Epoxide Hydrolases