Glycerophosphodiesterase (GpdQ) is a binuclear metallophosphatase that catalyzes the hydrolytic cleavage of mono-, di-, and triphosphoester bonds of a wide range of critical molecules. Upon substrate binding, this enzyme undergoes a complex transformation from an inactive mononuclear form (Em, where the metal resides in the α site) to an active binuclear center (Eb-S, with metals bound to both the α and β sites) through a mononuclear, substrate-bound intermediate state (Em-S). In this study, all-atom molecular dynamics simulations have been employed to investigate structures and dynamical transformations in this process using eight different variants, i.e., five wild-type and three mutant forms of the enzyme. Additionally, the effects of an actual substrate, bis-(para-nitrophenyl) phosphate (bpNPP), a metal-bridging nucleophilic hydroxyl, and specific first and second coordination shell residues have been investigated. The initial binding of the substrate to Em enhances the metal binding affinity of the α site and prepares the β site for coordination of the second metal ion. These results are in agreement with stopped-flow fluorescence and calorimetry data. In Eb-S, the computed increase in the substrate and metal (both α and β) binding energies is also in line with the experimental data. However, removal of the substrate from this complex is found to cause substantial reduction in binding energies of both α and β metals. The role of the substrate in the creation and stabilization of the active site predicted in this study is supported by the kinetic measurements using both stopped-flow and nuclear magnetic resonance techniques. Importantly, residue Asn80, a ligand of the metal in the β site, exhibits coordination flexibility by acting as a gate in the formation of Eb-S, in good agreement with mutagenesis and spectroscopic data.
ASJC Scopus subject areas
- Physical and Theoretical Chemistry
- Surfaces, Coatings and Films
- Materials Chemistry