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Theoretical Investigation of the Second-Shell Mechanism of Nitrile Hydratase
KTH, School of Biotechnology (BIO), Theoretical Chemistry.
KTH, School of Biotechnology (BIO), Theoretical Chemistry.
2008 (English)In: European Journal of Inorganic Chemistry, ISSN 1434-1948, E-ISSN 1099-0682, no 9, 1406-1412 p.Article in journal (Refereed) Published
Abstract [en]

Nitrile hydratases (NHases) are biocatalytically important enzymes that are utilized in the industrial production of acrylamide and nicotinamide. There are two different classes of NHases, harbouring either a low-spin Fe-III or a low-spin Co-III ion in the active site, in each case with the same peculiar set of ligands, involving deprotonated backbone amides and oxidized cysteine residues. The detailed reaction mechanism of NHase has not been established yet, but different proposals have been put forward. Depending on the binding site of the substrate, these can be divided into first-shell and second-shell mechanisms, respectively, Recently, we have investigated different first-shell mechanisms using quantum-chemical active-site models based on the iron-dependent NHase (Inorg. Chem. 2007, 46, 4850). Here we continue our investigation of the NHase reaction by exploring two different variations of the second-shell mechanism of the iron-dependent NHase. In the first, a metal-bound hydroxide ion performs a nucleophilic attack on the nitrile substrate, while in the second investigated mechanism, the oxidized cysteine, Cys114-SO-, acts as the nucleophile. We report energies, optimized transition state, and intermediate geometries for both investigated mechanisms. The calculated barriers are similar to the previously reported first-shell mechanism involving Cys114-SO- as catalytic base.

Place, publisher, year, edition, pages
2008. no 9, 1406-1412 p.
Keyword [en]
nitrile hydratase; enzyme catalysis; density functional theory; reaction mechanism
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:kth:diva-8109DOI: 10.1002/ejic.200701137ISI: 000254555300007Scopus ID: 2-s2.0-53249107748OAI: oai:DiVA.org:kth-8109DiVA: diva2:13340
Note
QC 20100811. Uppdaterad från in press till published (20100811).Available from: 2008-03-18 Created: 2008-03-18 Last updated: 2010-08-11Bibliographically approved
In thesis
1. Nitrile Hydratases and Epoxide-Transforming Enzymes: Quantum Chemical Modeling of Reaction Mechanisms and Selectivities
Open this publication in new window or tab >>Nitrile Hydratases and Epoxide-Transforming Enzymes: Quantum Chemical Modeling of Reaction Mechanisms and Selectivities
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Quantum chemical studies of enzymatic reactions are able to provide detailed insight into mechanisms and catalytic strategies. The energetic feasibility of proposed mechanisms can be established, and new possible reaction pathways can be put forward. The role of the involved active site residues can be analyzed in detail and the origins for experimentally observed selectivities can be investigated. Density functional theory (DFT), in particular the hybrid functional B3LYP, is the method of choice in this kind of studies.

In this thesis, the reaction mechanisms of several enzymes have been explored using the B3LYP functional. The studied enzymes include limonene epoxide hydrolase (LEH), soluble epoxide hydrolase (sEH), haloalcohol dehalogenase (HheC), and nitrile hydratase (NHase). Transition states and intermediates along various reaction pathways were optimized and evaluated.

For the three epoxide-transforming enzymes, the role of the proposed catalytic residues could be confirmed. Analysis of in silico mutations helped to quantify the effect of various functional groups on the barriers and regioselectivities of epoxide opening. A detailed analysis of the factors governing the enzymatic regioselectivities is given.

For nitrile hydratase, various putative first- and second-shell mechanisms have been studied. Active site models based on both the Co(III)-NHase and the Fe(III)-NHase were employed. The studied mechanisms include general base-catalyzed reaction pathways with water as nucleophile as well as two pathways involving cysteine-sulfenate as nucleophile. Several computed mechanisms exhibit similar barriers, making it difficult to pinpoint the true NHase mechanism.

Place, publisher, year, edition, pages
Stockholm: KTH, 2008. x, 74 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2008:1
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-4668 (URN)978-91-7178-885-6 (ISBN)
Public defence
2008-04-04, FB52, AlbaNova, Roslagstullsbacken 21, Stockholm, 10:30
Opponent
Supervisors
Note
QC 20100811Available from: 2008-03-18 Created: 2008-03-18 Last updated: 2010-08-11Bibliographically approved

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