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Quantum Chemical Modeling of Binuclear Zinc Enzymes
KTH, School of Biotechnology (BIO), Theoretical Chemistry.
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 [en]
Quantum Chemical Modeling, Binuclear, Zinc, Enzyme, DFT, Mechanism
National Category
Theoretical Chemistry
Identifiers
URN: urn:nbn:se:kth:diva-9705ISBN: 978-91-7415-173-2 (print)OAI: oai:DiVA.org:kth-9705DiVA: diva2:127039
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
List of papers
1. Theoretical Study of the Phosphotriesterase Reaction Mechanism
Open this publication in new window or tab >>Theoretical Study of the Phosphotriesterase Reaction Mechanism
2007 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 111, no 6, 1253-1255 p.Article in journal (Refereed) Published
Abstract [en]

Phosphotriesterase (PTE) is a binuclear zinc enzyme that catalyzes the hydrolysis of extremely toxic organophosphate triesters. In the present work, we have investigated the reaction mechanism of PTE using the hybrid density functional theory method B3LYP. We present a potential energy surface for the reaction and provide characterization of the transition states and intermediates. We used the high resolution crystal structure to construct a model of the active site of PTE, containing the two zinc ions and their first shell ligands. The calculations provide strong support to an associative mechanism for the hydrolysis of phosphotriesters by PTE. No protonation of the leaving group was found to be necessary. In particular, the calculations demonstrate that the nucleophilicity of the bridging hydroxide is sufficient to be utilized in the hydrolysis reaction, a feature that is of importance for a number of other di-zinc enzymes.

Keyword
BACTERIAL PHOSPHOTRIESTERASE; ACTIVE-SITE; 3-DIMENSIONAL STRUCTURE; PSEUDOMONAS-DIMINUTA; DENSITY; HYDROLYSIS; ENERGY; MODEL; BINUCLEAR; ALKALINE
National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-9747 (URN)10.1021/jp068500n (DOI)000244039700002 ()2-s2.0-33847410577 (Scopus ID)
Note
QC 20100714Available from: 2008-12-05 Created: 2008-12-05 Last updated: 2017-12-14Bibliographically approved
2. Technical aspects of quantum chemical modeling of enzymatic reactions: the case of phosphotriesterase
Open this publication in new window or tab >>Technical aspects of quantum chemical modeling of enzymatic reactions: the case of phosphotriesterase
2008 (English)In: Theoretical Chemistry accounts, ISSN 1432-881X, E-ISSN 1432-2234, Vol. 120, no 4-6, 515-522 p.Article in journal (Refereed) Published
Abstract [en]

Quantum chemical methods are today a powerful tool in the study of enzymatic reaction mechanisms. In this paper we evaluate the adequacy of some of the technical approximations frequently used in the modeling of enzyme reactions with high level methods. These include the choice of basis set for geometry optimizations and energy evaluation, the choice of dielectric constant to model the enzyme surrounding, and the effects of locking the centers of truncation. As a test case, we choose the phosphotriesterase enzyme, which is a binuclear zinc enzyme that catalyzes the hydrolysis of organophosphate triesters.

Keyword
SOLUBLE EPOXIDE HYDROLASE; REACTION-MECHANISM; BACTERIAL PHOSPHOTRIESTERASE; RIBONUCLEOTIDE REDUCTASE; PSEUDOMONAS-DIMINUTA; METHYL TRANSFER; WARFARE AGENTS; ACTIVE-SITE; DENSITY; ENZYMES
National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-9748 (URN)10.1007/s00214-008-0430-y (DOI)000256765900022 ()2-s2.0-45449084623 (Scopus ID)
Note
QC 20100714Available from: 2008-12-05 Created: 2008-12-05 Last updated: 2017-12-14Bibliographically approved
3. Structure of Diethyl Phosphate Bound to the Binuclear Metal Center of Phosphotriesterase
Open this publication in new window or tab >>Structure of Diethyl Phosphate Bound to the Binuclear Metal Center of Phosphotriesterase
Show others...
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.

Keyword
BACTERIAL PHOSPHOTRIESTERASE; PSEUDOMONAS-DIMINUTA; ISOASPARTYL DIPEPTIDASE; REACTION-MECHANISM; ESCHERICHIA-COLI; D-AMINOACYLASE; HYDROLYSIS; DENSITY; ENZYME; ENERGY
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-9749 (URN)10.1021/bi800971v (DOI)000258866700015 ()2-s2.0-51549089373 (Scopus ID)
Note
QC 20100714Available from: 2008-12-05 Created: 2008-12-05 Last updated: 2017-12-14Bibliographically approved
4. Peptide Hydrolysis by the Binuclear Zinc Enzyme Aminopeptidase from Aeromonas proteolytica: A Density Functional Theory Study
Open this publication in new window or tab >>Peptide Hydrolysis by the Binuclear Zinc Enzyme Aminopeptidase from Aeromonas proteolytica: A Density Functional Theory Study
Show others...
2008 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 112, no 8, 2494-2500 p.Article in journal (Refereed) Published
Abstract [en]

Aminopeptidase from Aeromonas proteolytica (AAP) is a binuclear zinc enzyme that catalyzes the cleavage of the N-terminal amino acid residue of peptides and proteins. In this study, we used density functional methods to investigate the reaction mechanism of this enzyme. A model of the active site was constructed on the basis of the X-ray crystal structure of the native enzyme, and a model dipeptide was used as a substrate. It was concluded that the hydroxide is capable of performing a nucleophilic attack at the peptide carbonyl from its bridging position without the need to first become terminal. The two zinc ions are shown to have quite different roles. Zn2 binds the amino group of the substrate, thereby orienting it toward the nucleophile, while Zn1 stabilizes the alkoxide ion of the tetrahedral intermediate, thereby lowering the barrier for the nucleophilic attack. The rate-limiting step is suggested to be the protonation of the nitrogen of the former peptide bond, which eventually leads to the cleavage of the C−N bond.

Keyword
L-LEUCINEPHOSPHONIC ACID; PROTEOLYTICA AMINOPEPTIDASE; CRYSTALLOGRAPHIC CHARACTERIZATION; SUBSTRATE-BINDING; CRYSTAL-STRUCTURE; TRANSITION-STATE; IONIC-SOLUTIONS; AB-INITIO; MECHANISM; INHIBITION
National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-9750 (URN)10.1021/jp710035j (DOI)000253355500023 ()2-s2.0-40549100037 (Scopus ID)
Note
QC 20100714Available from: 2008-12-08 Created: 2008-12-08 Last updated: 2017-12-14Bibliographically approved
5. Reaction mechanism of the binuclear zinc enzyme glyoxalase II: A theoretical study
Open this publication in new window or tab >>Reaction mechanism of the binuclear zinc enzyme glyoxalase II: A theoretical study
2009 (English)In: Journal of Inorganic Biochemistry, ISSN 0162-0134, E-ISSN 1873-3344, Vol. 103, no 2, 274-281 p.Article in journal (Refereed) Published
Abstract [en]

The glyoxalase system catalyzes the conversion of toxic methylglyoxal to nontoxic d-lactic acid using glutathione (GSH) as a coenzyme. Glyoxalase II (GlxII) is a binuclear Zn enzyme that catalyzes the second step of this conversion, namely the hydrolysis of S-d-lactoylglutathione, which is the product of the Glyoxalase I (GlxI) reaction. In this paper we use density functional theory method to investigate the reaction mechanism of GlxII. A model of the active site is constructed on the basis of the X-ray crystal structure of the native enzyme. Stationary points along the reaction pathway are optimized and the potential energy surface for the reaction is calculated. The calculations give strong support to the previously proposed mechanism. It is found that the bridging hydroxide is capable of performing nucleophilic attack at the substrate carbonyl to form a tetrahedral intermediate. This step is followed by a proton transfer from the bridging oxygen to Asp58 and finally C–S bond cleavage. The roles of the two zinc ions in the reaction mechanism are analyzed. Zn2 is found to stabilize the charge of tetrahedral intermediate thereby lowering the barrier for the nucleophilic attack, while Zn1 stabilizes the charge of the thiolate product, thereby facilitating the C–S bond cleavage. Finally, the energies involved in the product release and active-site regeneration are estimated and a new possible mechanism is suggested.

Keyword
BETA-LACTAMASE; CRYSTAL-STRUCTURE; BACTERIAL PHOSPHOTRIESTERASE; BACTEROIDES-FRAGILIS; CATALYTIC MECHANISM; IN-VITRO; DENSITY; BINDING; METHYLGLYOXAL; HYDROLYSIS
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-9751 (URN)10.1016/j.jinorgbio.2008.10.016 (DOI)000262919700015 ()2-s2.0-58149492560 (Scopus ID)
Note
QC 20100714. Uppdaterad från in press till published (20100714).Available from: 2008-12-08 Created: 2008-12-08 Last updated: 2017-12-14Bibliographically approved
6. Insights into the Transition States and Mechanism of Alkaline Phosphatase Reaction from the DFT Calculations
Open this publication in new window or tab >>Insights into the Transition States and Mechanism of Alkaline Phosphatase Reaction from the DFT Calculations
(English)Manuscript (Other academic)
National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-9752 (URN)
Note
QC 20100715Available from: 2008-12-08 Created: 2008-12-08 Last updated: 2010-07-15Bibliographically approved

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