Structure of Diethyl Phosphate Bound to the Binuclear Metal Center of Phosphotriesterase

Kim, J; Tsai, P; Chen, S; Himo, F; Almo, S; Raushel, F

doi:10.1021/bi800971v

Title: Structure of Diethyl Phosphate Bound to the Binuclear Metal Center of Phosphotriesterase

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Abstract

The bacterial phosphotriesterase (PTE) from Pseudomonas diminuta catalyzes the hydrolysis of organophosphate esters at rates close to the diffusion limit. X-ray diffraction studies have shown that a binuclear metal center is positioned in the active site of PTE and that this complex is responsible for the activation of the nucleophilic water from solvent. In this paper, the three-dimensional structure of PTE was determined in the presence of the hydrolysis product, diethyl phosphate (DEP), and a product analogue, cacodylate. In the structure of the PTE-diethyl phosphate complex, the DEP product is found symmetrically bridging the two divalent cations. The DEP displaces the hydroxide from solvent that normally bridges the two divalent cations in structures determined in the presence or absence of substrate analogues. One of the phosphoryl oxygen atoms in the PTE-DEP complex is 2.0 Angstroms from the a-metal ion, while the other oxygen is 2.2 Angstroms from the {beta}-metal ion. The two metal ions are separated by a distance of 4.0 Angstroms. A similar structure is observed in the presence of cacodylate. Analogous complexes have previously been observed for the product complexes of isoaspartyl dipeptidase, d-aminoacylase, and dihydroorotase from the amidohydrolase superfamily of enzymes. The experimentally determined structure of themore »« less

Authors:: Kim, J; Tsai, P; Chen, S; Himo, F; Almo, S; Raushel, F

Publication Date:: Tue Jan 01 00:00:00 EST 2008

Research Org.:: Brookhaven National Lab. (BNL), Upton, NY (United States). National Synchrotron Light Source

Sponsoring Org.:: Doe - Office Of Science

OSTI Identifier:: 959828

Report Number(s):: BNL-82814-2009-JA
Journal ID: ISSN 0006-2960; BICHAW; TRN: US1005813

DOE Contract Number:: DE-AC02-98CH10886

Resource Type:: Journal Article

Journal Name:: Biochemistry

Additional Journal Information:: Journal Volume: 47; Journal Issue: 36; Journal ID: ISSN 0006-2960

Country of Publication:: United States

Language:: English

Subject:: 72 PHYSICS OF ELEMENTARY PARTICLES AND FIELDS; ATOMS; CATIONS; DIFFUSION; ENZYMES; ESTERS; FUNCTIONALS; HYDROLYSIS; HYDROXIDES; OXYGEN; PHOSPHATES; PROTONS; PSEUDOMONAS; REACTION KINETICS; SOLVENTS; SUBSTRATES; WATER; X-RAY DIFFRACTION; national synchrotron light source

Citation Formats


                    Kim, J, Tsai, P, Chen, S, Himo, F, Almo, S, and Raushel, F. Structure of Diethyl Phosphate Bound to the Binuclear Metal Center of Phosphotriesterase.  United States: N. p., 2008. 
        Web.  doi:10.1021/bi800971v.

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                    Kim, J, Tsai, P, Chen, S, Himo, F, Almo, S, & Raushel, F. Structure of Diethyl Phosphate Bound to the Binuclear Metal Center of Phosphotriesterase.  United States.  https://doi.org/10.1021/bi800971v

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                    Kim, J, Tsai, P, Chen, S, Himo, F, Almo, S, and Raushel, F. 2008.  
        "Structure of Diethyl Phosphate Bound to the Binuclear Metal Center of Phosphotriesterase".  United States.  https://doi.org/10.1021/bi800971v.

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@article{osti_959828,

  title        = {Structure of Diethyl Phosphate Bound to the Binuclear Metal Center of Phosphotriesterase},

  author       = {Kim, J and Tsai, P and Chen, S and Himo, F and Almo, S and Raushel, F},

  abstractNote = {The bacterial phosphotriesterase (PTE) from Pseudomonas diminuta catalyzes the hydrolysis of organophosphate esters at rates close to the diffusion limit. X-ray diffraction studies have shown that a binuclear metal center is positioned in the active site of PTE and that this complex is responsible for the activation of the nucleophilic water from solvent. In this paper, the three-dimensional structure of PTE was determined in the presence of the hydrolysis product, diethyl phosphate (DEP), and a product analogue, cacodylate. In the structure of the PTE-diethyl phosphate complex, the DEP product is found symmetrically bridging the two divalent cations. The DEP displaces the hydroxide from solvent that normally bridges the two divalent cations in structures determined in the presence or absence of substrate analogues. One of the phosphoryl oxygen atoms in the PTE-DEP complex is 2.0 Angstroms from the a-metal ion, while the other oxygen is 2.2 Angstroms from the {beta}-metal ion. The two metal ions are separated by a distance of 4.0 Angstroms. A similar structure is observed in the presence of cacodylate. Analogous complexes have previously been observed for the product complexes of isoaspartyl dipeptidase, d-aminoacylase, and dihydroorotase from the amidohydrolase superfamily of enzymes. The experimentally determined structure of the PTE-diethyl phosphate product complex is inconsistent with a recent proposal based upon quantum mechanical/molecular mechanical simulations which postulated the formation of an asymmetrical product complex bound exclusively to the {beta}-metal ion with a metal-metal separation of 5.3 Angstroms. This structure is also inconsistent with a chemical mechanism for substrate hydrolysis that utilizes the bridging hydroxide as a base to abstract a proton from a water molecule loosely associated with the a-metal ion. Density functional theory (DFT) calculations support a reaction mechanism that utilizes the bridging hydroxide as the direct nucleophile in the hydrolysis of organophosphate esters by PTE.},

  doi          = {10.1021/bi800971v},

  url          = {https://www.osti.gov/biblio/959828},
  journal      = {Biochemistry},

  issn         = {0006-2960},

  number       = 36,

  volume       = 47,

  place        = {United States},

  year         = {Tue Jan 01 00:00:00 EST 2008},

  month        = {Tue Jan 01 00:00:00 EST 2008}

}

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