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Temperature and pH dependence of enzyme catalyzed hydrolysis of trans-methylstyrene oxide: a unifying kinetic model for observed hysteresis, cooperativity and regioselectivity
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
2010 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 49, no 10, 2297-2304 p.Article in journal (Refereed) Published
Abstract [en]

The underlying enzyme kinetics behind the regioselective promiscuity shown by epoxide hydrolases towards certain epoxides has been studied. The effects of temperature and pH on regioselectivity was investigated by analyzing the stereochemistry of hydrolysis products of (1R,2R)-trans-2-methylstyrene oxide between 14 to 46 °C and pH 6.0 to 9.0, either catalyzed by the potato epoxide hydrolase StEH1 or in the absence of enzyme. In the enzyme-catalyzed reaction, a switch of preferred epoxide carbon that is subjected to nucleophilic attack is observed at pH values above 8. The enzyme also displays cooperativity in substrate saturation plots when assayed at temperatures ≤30 °C and at intermediate pH. The cooperativity is lost at higher assay temperatures. Cooperativity can originate from a kinetic mechanism involving hysteresis and will be dependent on the relationship between kcat and the rate of interconversion between two different Michaelis complexes. In the case of the studied reactions, the proposed different Michaelis complexes are enzyme-substrate complexes in which the epoxide substrate is bound in different binding modes, allowing for separate pathways towards product formation. The assumption of separated, but interacting, reaction pathways is supported by that formation of the two product enantiomers also display distinct pH dependencies of kcat/KM. The thermodynamic parameters describing the differences in activation enthalpy and entropy suggest that 1) regioselectivity is primarily dictated by differences in activation entropy with positive values of both ΔΔH and ΔΔS, and 2) the hysteretic behavior is linked to an interconversion between Michaelis complexes with rates increasing with temperature. From the collected data, we propose that hysteresis, regioselectivity and, when applicable, hysteretic cooperativity are closely linked properties, explained by the kinetic mechanism earlier introduced by our group.

Place, publisher, year, edition, pages
2010. Vol. 49, no 10, 2297-2304 p.
Keyword [en]
epoxide hydrolase, regioselectivity, hysteresis, StEH1
National Category
Chemical Sciences
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:uu:diva-112272DOI: 10.1021/bi902157bISI: 000275227000022PubMedID: 20146441OAI: oai:DiVA.org:uu-112272DiVA: diva2:285617
Available from: 2010-01-12 Created: 2010-01-12 Last updated: 2017-12-12Bibliographically approved
In thesis
1. Exploring Selectivity and Hysteresis: Kinetic Studies on a Potato Epoxide Hydrolase
Open this publication in new window or tab >>Exploring Selectivity and Hysteresis: Kinetic Studies on a Potato Epoxide Hydrolase
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The kinetic mechanism of an α/β hydrolase fold epoxide hydrolase from potato, StEH1, has been studied with the aims of explaining the underlying causes for enantio- and regioselectivity, both being important for product purity. Further effort has been laid upon understanding the causes of a hysteretic behavior discovered in the measurements leading to Paper I.

The enantioselectivity was investigated with substrates differing only in substituent size at one carbon of the oxirane ring structure. In catalysis with trans-stilbene oxide and styrene oxide, enantioselectivity is the result of differences in alkylation rates.

In pre-steady state measurement with trans-2-methylstyrene oxide (2-MeSO), a rate-limiting step involving slow transitions, referred to as hysteresis, was discovered. With this substrate enantioselectivity is proposed to be a consequence of the catalytic rate of (1R,2R)-enantiomer being more influenced by the hysteretic behavior than was the rate of the other enantiomer.

In steady-state measurements with (1R,2R)-2-MeSO, at different temperatures and pH, hysteretic cooperativity was displayed. It can be concluded that this behavior is dependent on the relationship between kcat and the rate of transition between two Michaelis complexes. From the differences in pH dependence of kcat/KM in formation of the two diols resulting from low regioselectivity in catalysis of (1R,2R)-2-MeSO, it is suggested that hysteresis is a result of the substrates placed in different conformational modes within the active site cavity.

Regioselectivity is proposed to be the result of specific interactions between the catalytically important Tyr and the substrate, with a link between KM-values and degree of regioselectivity. Furthermore, the hysteretic kinetic model proposed can explain hysteresis, cooperativity and regioselectivity resulting from StEH1 catalyzed hydrolysis of (1R,2R)-2-MeSO.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2010. 62 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 707
Keyword
Epoxide hydrolase; regioselectivity; selectivity; hysteresis; conformational changes; cooperativity; deep eutectic solvents; methylstyrene oxide
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-112285 (URN)978-91-554-7701-1 (ISBN)
Public defence
2010-02-18, B41, BMC, Husargatan 3, Uppsala, 10:15 (English)
Opponent
Supervisors
Available from: 2010-01-28 Created: 2010-01-12 Last updated: 2010-01-28Bibliographically approved

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