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Theoretical investigation of the reaction mechanism of the dinuclear zinc enzyme dihydroorotase
KTH, School of Biotechnology (BIO), Theoretical Chemistry.
KTH, School of Biotechnology (BIO), Theoretical Chemistry.
2008 (English)In: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 14, no 14, 4287-4292 p.Article in journal (Refereed) Published
Abstract [en]

The reaction mechanism of the dinuclear zinc enzyme dihydroorotase was investigated by using hybrid density functional theory. This enzyme catalyzes the reversible interconversion of dihydroorotate and carbamoyl aspartate. Two reaction mechanisms in which the important active site residue Asp250 was either protonated or unprotonated were considered. The calculations establish that Asp250 must be unprotonated for the reaction to take place. The bridging hydroxide is shown to be capable of performing nucleophilic attack on the substrate from its bridging position and the role of Znβ is argued to be the stabilization of the tetrahedral intermediate and the transition state leading to it, thereby lowering the barrier for the nucleophilic attack. It is furthermore concluded that the rate-limiting step is the protonation of the amide nitrogen by Asp250 coupled with C-N bond cleavage, which is consistent with previous experimental findings from isotope labeling studies.

Place, publisher, year, edition, pages
2008. Vol. 14, no 14, 4287-4292 p.
Keyword [en]
Density functional calculations, Dihydroorotase, Enzyme catalysis, Reaction mechanisms
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:kth:diva-10171DOI: 10.1002/chem.200701948ISI: 000256131800018Scopus ID: 2-s2.0-46749153927OAI: oai:DiVA.org:kth-10171DiVA: diva2:209843
Note
QC 20101004Available from: 2009-03-27 Created: 2009-03-27 Last updated: 2010-10-04Bibliographically approved
In thesis
1. Mechanistic insights into dinuclear zinc enzymes from density functional theory studies
Open this publication in new window or tab >>Mechanistic insights into dinuclear zinc enzymes from density functional theory studies
2009 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

In this thesis, quantum chemical methods have been used to shed light on the reaction mechanisms of several dinuclear zinc enzymes. The enzymes studied are involved in the hydrolysis of phosphates, amides, and carboxylic esters, namely RNase Z, Dihydroorotase (DHO), and N-acyl homoserine lactone hydrolase (AHL lactonase). The density functional method B3LYP, together with quite large active site models, was used to investigate these enzymatic reactions. Several plausible proposed mechanisms, involving protonation states of important active site residues (DHO), substrate orientations (AHL lactonase), have been considered. The calculated energetics can be used to assess the feasibility of the suggested reaction mechanisms. Based on the calculations and also on other related dinuclear zinc enzymes studied previously, some general mechanistic features have been uncovered.

For all three enzymes, the nucleophilicity of the bridging hydroxide is shown to be sufficient to perform the nucleophilic attack on the substrates. During the attack, the negative charge is transferred from the bridging hydroxide to the substrate oxygen (P=O or C=O). For phosphate hydrolysis, an in line attack have been suggested for RNase Z. In addition, the two zinc ions in RNase Z are directly involved in stabilizing the negative charge in the penta-coordinated transition states. For carbonyl substrates, only one zinc ion participates in the oxygen anion stabilization in the transition states and the tetrahedral intermediates. Furthermore, the enzymes always use the zinc ion with less negatively-charged ligands to play such role.

All the substrates investigated have poor leaving groups. Therefore, either the zinc ions or some active site residues help the cleavage of the scissile bond. For RNase Z, a Glu-His diad was suggested to protonate the leaving group. For DHO, an Asp residue was shown to transfer a proton from the bridging hydroxide to the leaving group nitrogen. For AHL lactonase, a zinc ion was also observed to stabilize the leaving oxygen anion.

Place, publisher, year, edition, pages
Stockholm: KTH, 2009. 59 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2009:4
National Category
Other Basic Medicine
Identifiers
urn:nbn:se:kth:diva-10175 (URN)978-91-7415-261-6 (ISBN)
Presentation
(English)
Available from: 2009-03-27 Created: 2009-03-27 Last updated: 2010-10-18Bibliographically approved

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