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Mechanistic insights into dinuclear zinc enzymes from density functional theory studies
KTH, School of Biotechnology (BIO), Theoretical Chemistry.
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: urn:nbn:se:kth:diva-10175ISBN: 978-91-7415-261-6 (print)OAI: oai:DiVA.org:kth-10175DiVA: diva2:209857
Presentation
(English)
Available from: 2009-03-27 Created: 2009-03-27 Last updated: 2010-10-18Bibliographically approved
List of papers
1. Theoretical investigation of the reaction mechanism of the dinuclear zinc enzyme dihydroorotase
Open this publication in new window or tab >>Theoretical investigation of the reaction mechanism of the dinuclear zinc enzyme dihydroorotase
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.

Keyword
Density functional calculations, Dihydroorotase, Enzyme catalysis, Reaction mechanisms
National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-10171 (URN)10.1002/chem.200701948 (DOI)000256131800018 ()2-s2.0-46749153927 (Scopus ID)
Note
QC 20101004Available from: 2009-03-27 Created: 2009-03-27 Last updated: 2017-12-13Bibliographically approved
2. Reaction mechanism of the dinuclear zinc enzyme N-acyl-L-homoserine lactone hydrolase: a quantum chemical study
Open this publication in new window or tab >>Reaction mechanism of the dinuclear zinc enzyme N-acyl-L-homoserine lactone hydrolase: a quantum chemical study
2009 (English)In: Inorganic Chemistry, ISSN 0020-1669, E-ISSN 1520-510X, Vol. 48, no 4, 1442-1448 p.Article in journal (Refereed) Published
Abstract [en]

N-acyl-L-homoserine lactone hydrolase (AHL lactonase) is a dinuclear zinc enzyme responsible for the hydrolytic ring opening of AHLs, disrupting quorum sensing in bacteria. The reaction mechanism is investigated using hybrid density functional theory. A model of the active site is designed on the basis of the X-ray crystal structure, and stationary points along the reaction pathway are optimized and analyzed. Two possible mechanisms based on two different substrate orientations are considered. The calculations give support to a reaction mechanism that involves two major chemical steps: nucleophilic attack on the substrate carbonyl carbon by the bridging hydroxide and ring opening by direct ester C - O bond cleavage. The roles of the two zinc ions are analyzed. Zn1 is demonstrated to stabilize the charge of the tetrahedral intermediate, thereby facilitating the nucleophilic attack, while Zn2 stabilizes the charge of the alkoxide resulting from the ring opening, thereby lowering the barrier for the C - O bond cleavage.

Keyword
QUORUM-QUENCHING LACTONASE, BACILLUS-THURINGIENSIS, IDENTIFICATION, SPECIFICITY, BACTERIA, DENSITY, ENERGY, AIIA, COMMUNICATION, VIRULENCE
National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-10172 (URN)10.1021/ic801531n (DOI)000263227100026 ()2-s2.0-64349108338 (Scopus ID)
Note
QC 20100928Available from: 2009-03-27 Created: 2009-03-27 Last updated: 2017-12-13Bibliographically approved
3. Theoretical study of the RNA hydrolysis mechanism of the dinuclear zinc enzyme RNase Z
Open this publication in new window or tab >>Theoretical study of the RNA hydrolysis mechanism of the dinuclear zinc enzyme RNase Z
2009 (English)In: European Journal of Inorganic Chemistry, ISSN 1434-1948, E-ISSN 1099-1948, no 20, 2967-2972 p.Article in journal (Refereed) Published
Abstract [en]

 RNase Z is a dinuclear zinc enzyme that catalyzes the removal of the tRNA 3'-end trailer. Density functional theory is used to investigate the phosphodiester hydrolysis mechanism of this enzyme with a model of the active site constructed on the basis of the crystal structure. The calculations imply that the reaction proceeds through two steps. The first step is a nucleophihc attack by a bridging hydroxide coupled with protonation of the leaving group by a Glu-His diad. Subsequently, a water molecule activated by the same Glu-His diad makes a reverse attack, regenerating the bridging hydroxide. The second step is calculated to be the rate-limiting step with a barrier of 18 kcal/mol, in good agreement with experimental kinetic studies. Both zinc ions participate in substrate binding and orientation, facilitating nucleophilic attack. In addition, they act as electrophilic catalysts to stabilize the pentacoordinate trigonal-bipyramidal transition states.

Keyword
Enzyme catalysis, Metalloenzymes, Dinuclear zinc enzymes, Density functional calculations, Hydrolysis, Reaction mechanisms, Zinc
National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-10174 (URN)10.1002/ejic.200900202 (DOI)000268290000010 ()
Note
QC 20101018. Uppdaterad från Submitted till Published (20101018).Available from: 2009-03-27 Created: 2009-03-27 Last updated: 2017-12-13Bibliographically approved

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