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Reaction mechanism of the binuclear zinc enzyme glyoxalase II: A theoretical study
KTH, School of Biotechnology (BIO), Theoretical Chemistry.
Beijing Normal Univ, Coll Chem.
KTH, School of Biotechnology (BIO), Theoretical Chemistry.
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.

Place, publisher, year, edition, pages
2009. Vol. 103, no 2, 274-281 p.
Keyword [en]
BETA-LACTAMASE; CRYSTAL-STRUCTURE; BACTERIAL PHOSPHOTRIESTERASE; BACTEROIDES-FRAGILIS; CATALYTIC MECHANISM; IN-VITRO; DENSITY; BINDING; METHYLGLYOXAL; HYDROLYSIS
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-9751DOI: 10.1016/j.jinorgbio.2008.10.016ISI: 000262919700015Scopus ID: 2-s2.0-58149492560OAI: oai:DiVA.org:kth-9751DiVA: diva2:127478
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
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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