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Structure of Diethyl Phosphate Bound to the Binuclear Metal Center of Phosphotriesterase
Albert Einstein Coll Med.
Texas A&M Univ, Dept Chem.
KTH, School of Biotechnology (BIO), Theoretical Chemistry.
KTH, School of Biotechnology (BIO), Theoretical Chemistry.
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2008 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 47, no 36, 9497-9504 p.Article in journal (Refereed) Published
Abstract [en]

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 Å from the α-metal ion, while the other oxygen is 2.2 Å from the β-metal ion. The two metal ions are separated by a distance of 4.0 Å. 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 β-metal ion with a metal−metal separation of 5.3 Å. 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 α-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.

Place, publisher, year, edition, pages
2008. Vol. 47, no 36, 9497-9504 p.
Keyword [en]
BACTERIAL PHOSPHOTRIESTERASE; PSEUDOMONAS-DIMINUTA; ISOASPARTYL DIPEPTIDASE; REACTION-MECHANISM; ESCHERICHIA-COLI; D-AMINOACYLASE; HYDROLYSIS; DENSITY; ENZYME; ENERGY
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-9749DOI: 10.1021/bi800971vISI: 000258866700015Scopus ID: 2-s2.0-51549089373OAI: oai:DiVA.org:kth-9749DiVA: diva2:127410
Note
QC 20100714Available from: 2008-12-05 Created: 2008-12-05 Last updated: 2010-07-14Bibliographically approved
In thesis
1. Quantum Chemical Modeling of Binuclear Zinc Enzymes
Open this publication in new window or tab >>Quantum Chemical Modeling of Binuclear Zinc Enzymes
2008 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

In the present thesis, the reaction mechanisms of several di-zinc hydrolases have been explored using quantum chemical modeling of the enzyme active sites. The studied enzymes are phosphotriesterase (PTE), aminopeptidase from Aeromonas proteolytica (AAP), glyoxalase II (GlxII), and alkaline phosphatase (AP). All of them contain a binuclear divalent zinc core in the active site. The density functional theory (DFT) method B3LYP functional was employed in the investigations. The potential energy surfaces (PESs) for various reaction pathways have been mapped and the involved transition states and intermediates have been characterized. The hydrolyses of different types of substrates were examined, including phosphate esters (PTE and AP) and the substrates containing carbonyl group (AAP and GlxII). The roles of zinc ions and individual active-site residues were analyzed and general features of di-zinc enzymes have been characterized.

The bridging hydroxide stabilized by two zinc ions has been confirmed to be capable of the nucleophile in the hydrolysis reactions. PTE, AAP, and GlxII all employ the bridging hydroxide as the direct nucleophile. Furthermore, it is shown that either one of or both zinc ions provide the main catalytic power by stabilizing the negative charge developing during the reaction and thereby lowering the barriers. In the cases of GlxII and AP, one of zinc ions also contributes to the catalysis by stabilizing the leaving group. These features perfectly satisfy the two requisites for the hydrolysis, i.e. sufficient nucleophilicity and stabilization of charge. A competing mechanism, in which the bridging hydroxide acts as a base, was shown to have significantly higher barrier in the case of PTE.

For phosphate hydrolysis reactions, it is important to characterize the nature of the transition states involved in the reactions. Associative mechanisms were observed for both PTE and AP. The former uses a step-wise associative pathway via a penta-coordinated intermediate, while the latter proceeds through a concerted associative path via penta-coordinated transition states.

Finally, with PTE as a test case, systematic evaluation of the computational performance of the quantum chemical modeling approach has been performed. This assessment, coupled with other results of this thesis, provide an effective demonstration of the usefulness and powerfulness of quantum chemical active-site modeling in the exploration of enzyme reaction mechanisms and in the characterization of the transition states involved.

Place, publisher, year, edition, pages
Stockholm: KTH, 2008. viii, 66 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2008:27
Keyword
Quantum Chemical Modeling, Binuclear, Zinc, Enzyme, DFT, Mechanism
National Category
Theoretical Chemistry
Identifiers
urn:nbn:se:kth:diva-9705 (URN)978-91-7415-173-2 (ISBN)
Public defence
2008-12-19, FB53, AlbaNova, Roslagstullsbacken 21, Stockholm, 14:00 (English)
Opponent
Supervisors
Projects
Quantum Chemical Modeling of Binuclear Zinc Enzymes
Note
QC 20100715Available from: 2008-12-05 Created: 2008-11-28 Last updated: 2010-07-15Bibliographically approved

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