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Quantum Chemical Cluster Modeling of Enzymatic Reactions
Stockholm University, Faculty of Science, Department of Organic Chemistry.
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The Quantum chemical cluster approach has been shown to be quite powerful and efficient in the modeling of enzyme active sites and reaction mechanisms. In this thesis, the reaction mechanisms of several enzymes have been investigated using the hybrid density functional B3LYP. The enzymes studied include four dinuclear zinc enzymes, namely dihydroorotase, N-acyl-homoserine lactone hydrolase, RNase Z, and human renal dipeptidase, two trinuclear zinc enzymes, namely phospholipase C and nuclease P1, two tungstoenzymes, namely formaldehyde ferredoxin oxidoreductase and acetylene hydratase, aspartate α-decarboxylase, and mycolic acid cyclopropane synthase. The potential energy profiles for various mechanistic scenarios have been calculated and analyzed. The role of the metal ions as well as important active site residues has been discussed.

  In the cluster approach, the effects of the parts of the enzyme that are not explicitly included in the model are taken into account using implicit solvation methods.

  For all six zinc-dependent enzymes studied, the di-zinc bridging hydroxide has been shown to be capable of performing nucleophilic attack on the substrate. In addition, one, two, or even all three zinc ions participate in the stabilization of the negative charge in the transition states and intermediates, thereby lowering the barriers.

  For the two tungstoenzymes, several different mechanistic scenarios have been considered to identify the energetically most feasible one. For both enzymes, new mechanisms are proposed.

  Finally, the mechanism of mycolic acid cyclopropane synthase has been shown to be a direct methyl transfer to the substrate double bond, followed by proton transfer to the bicarbonate.

  From the studies of these enzymes, we demonstrate that density functional calculations are able to solve mechanistic problems related to enzymatic reactions, and a wealth of new insight can be obtained.

Place, publisher, year, edition, pages
Stockholm: Department of Organic Chemistry, Stockholm University , 2010. , 76 p.
Keyword [en]
cluster approach, density functional theory, B3LYP, enzyme, mechanism, dinuclear, trinuclear, zinc, tungsten, hydrolysis, decarboxylation, formaldehyde oxidation, hydration, methyl transfer
National Category
Theoretical Chemistry
Research subject
Organic Chemistry
Identifiers
URN: urn:nbn:se:su:diva-43026ISBN: 978-91-7447-129-8 (print)OAI: oai:DiVA.org:su-43026DiVA: diva2:353152
Public defence
2010-10-27, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 13:00 (English)
Opponent
Supervisors
Available from: 2010-10-05 Created: 2010-09-24 Last updated: 2010-10-07Bibliographically 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 inter-conversion 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 Zn-beta 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:su:diva-43036 (URN)10.1002/chem.200701948 (DOI)
Available from: 2010-09-24 Created: 2010-09-24 Last updated: 2017-12-12Bibliographically 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-homosedne 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.

National Category
Chemical Sciences
Identifiers
urn:nbn:se:su:diva-43034 (URN)10.1021/ic801531n (DOI)
Available from: 2010-09-24 Created: 2010-09-24 Last updated: 2017-12-12Bibliographically 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, Vol. 2009, 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.

Place, publisher, year, edition, pages
Weinheim: Wiley-VCH Verlag GmbH & Co. KGaA, 2009
Keyword
Enzyme catalysis; Metalloenzymes; Dinuclear zinc enzymes; Density functional calculations; Hydrolysis; Reaction mechanisms; Zinc
National Category
Other Basic Medicine
Identifiers
urn:nbn:se:su:diva-31365 (URN)10.1002/ejic.200900202 (DOI)000268290000010 ()
Available from: 2009-11-11 Created: 2009-11-11 Last updated: 2017-12-12Bibliographically approved
4. Dipeptide hydrolysis by the dinuclear zinc enzyme human renal dipeptidase: Mechanistic insights from DFT calculations
Open this publication in new window or tab >>Dipeptide hydrolysis by the dinuclear zinc enzyme human renal dipeptidase: Mechanistic insights from DFT calculations
2010 (English)In: Journal of Inorganic Biochemistry, ISSN 0162-0134, E-ISSN 1873-3344, Vol. 104, no 1, 37-46 p.Article in journal (Refereed) Published
Abstract [en]

The reaction mechanism of the dinuclear zinc enzyme human renal dipeptidase is investigated using hybrid density functional theory. This enzyme catalyzes the hydrolysis of dipeptides and beta-lactam antibiotics. Two different protonation states in which the important active site residue Asp288 is either neutral or ionized were considered. In both cases, the bridging hydroxide is shown to be capable of performing the nucleophilic attack on the substrate carbonyl carbon from its bridging position, resulting in the formation of a tetrahedral intermediate. This step is followed by protonation of the dipeptide nitrogen, coupled with C-N bond cleavage. The calculations establish that both cases have quite feasible energy barriers. When the Asp288 is neutral, the hydrolytic reaction occurs with a large exothermicity. However, the reaction becomes very close to thermoneutral with an ionized Asp288. The two zinc ions are shown to play different roles in the reaction. Zn1 binds the amino group of the substrate, and Zn2 interacts with the carboxylate group of the substrate, helping in orienting it for the nucleophilic attack. In addition, Zn2 stabilizes the oxyanion of the tetrahedral intermediate, thereby facilitating the nucleophilic attack

Keyword
Dipeptidase; Dinuclear zinc enzymes; Reaction mechanism; Density functional theory
National Category
Chemical Sciences
Identifiers
urn:nbn:se:su:diva-43035 (URN)10.1016/j.jinorgbio.2009.09.025 (DOI)000272417300005 ()
Available from: 2010-09-24 Created: 2010-09-24 Last updated: 2017-12-12Bibliographically approved
5. Reaction Mechanism of the Trinuclear Zinc Enzyme Phospholipase C: A Density Functional Theory Study
Open this publication in new window or tab >>Reaction Mechanism of the Trinuclear Zinc Enzyme Phospholipase C: A Density Functional Theory Study
2010 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 114, no 7, 2533-2540 p.Article in journal (Refereed) Published
Abstract [en]

Phosphatidylcholine-preferring phospholipase C is a trinuclear zinc-dependent phosphodiesterase, catalyzing the hydrolysis of choline phospholipids. In the present study, density functional theory is used to investigate the reaction mechanism of this enzyme. Two possible mechanistic scenarios were considered with a model of the active site designed on the basis of the high resolution X-ray crystal structure of the native enzyme. The calculations show that a Zn1 and Zn3 bridging hydroxide rather than a Zn1 coordinated water molecule performs the nucleophilic attack on the phosphorus center. Simultaneously, Zn2 activates a water molecule to protonate the leaving group. In the following step, the newly generated Zn2 bound hydroxide makes the reverse attack, resulting in the regeneration of the bridging hydroxide. The first step is calculated to be rate-limiting with a barrier of 17.3 kcal/mol, in good agreement with experimental kinetic studies. The zinc ions are suggested to orient the substrate for nucleophilic attack and provide electrostatic stabilization to the dianionic penta-coordinated trigonal bipyramidal transition states, thereby lowering the barrier.

National Category
Chemical Sciences
Identifiers
urn:nbn:se:su:diva-43033 (URN)10.1021/jp910992f (DOI)000274578500026 ()
Available from: 2010-09-24 Created: 2010-09-24 Last updated: 2017-12-12Bibliographically approved

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