Formation of Catalytically Active Binuclear Center of Glycerophosphodiesterase: A Molecular Dynamics Study

Thomas J. Paul, Gerhard Schenk, Rajeev Prabhakar

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)5797-5808
Number of pages12
JournalJournal of Physical Chemistry B
Volume122
Issue number22
DOIs
StatePublished - Jun 7 2018

Fingerprint

Molecular Dynamics Simulation
Molecular dynamics
Metals
molecular dynamics
Substrates
metals
Binding energy
enzymes
Enzymes
binding energy
Catalytic Domain
mutagenesis
Mutagenesis
Calorimetry
glycerophosphodiester phosphodiesterase
Hydroxyl Radical
Metal ions
affinity
cleavage
metal ions

ASJC Scopus subject areas

  • Physical and Theoretical Chemistry
  • Surfaces, Coatings and Films
  • Materials Chemistry

Cite this

Formation of Catalytically Active Binuclear Center of Glycerophosphodiesterase : A Molecular Dynamics Study. / Paul, Thomas J.; Schenk, Gerhard; Prabhakar, Rajeev.

In: Journal of Physical Chemistry B, Vol. 122, No. 22, 07.06.2018, p. 5797-5808.

Research output: Contribution to journalArticle

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AB - 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.

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