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Substrate-dependent hysteretic behavior in StEH1-catalyzed hydrolysis of styrene oxide derivatives
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.
2008 (English)In: The FEBS Journal, ISSN 1742-464X, Vol. 275, no 24, 6309-6320 p.Article in journal (Refereed) Published
Abstract [en]

The substrate selectivity and enantioselectivity of Solanum tuberosum epoxide hydrolase 1 (StEH1) have been explored by steady-state and pre-steady-state measurements on a series of styrene oxide derivatives. A preference for the (S)- or (S,S)-enantiomers of styrene oxide, 2-methylstyrene oxide and trans-stilbene oxide was established, with E-values of 43, 160 and 2.9, respectively. Monitoring of the pre-steady-state phase of the reaction with (S,S)-2-methylstyrene oxide revealed two observed rates for alkylenzyme formation. The slower of these rates showed a negative substrate concentration dependence, as did the rate of alkylenzyme formation in the reaction with the (R,R)-enantiomer. Such kinetic behavior is indicative of an additional, off-pathway step in the mechanism, referred to as hysteresis. On the basis of these data, a kinetic mechanism that explains the kinetic behavior with all tested substrates transformed by this enzyme is proposed. Regioselectivity of StEH1 in the catalyzed hydrolysis of 2-methylstyrene oxide was determined by 13C-NMR spectroscopy of 18O-labeled diol products. The (S,S)-enantiomer is attacked exclusively at the C-1 epoxide carbon, whereas the (R,R)-enantiomer is attacked at either position at a ratio of 65 : 35 in favor of the C-1 carbon. On the basis of the results, we conclude that differences in efficiency in stabilization of the alkylenzyme intermediates by StEH1 are important for enantioselectivity with styrene oxide or trans-stilbene oxide as substrate. With 2-methylstyrene oxide, slow conformational changes in the enzyme also influence the catalytic efficiency.

Place, publisher, year, edition, pages
2008. Vol. 275, no 24, 6309-6320 p.
National Category
Chemical Sciences
URN: urn:nbn:se:uu:diva-86664DOI: 10.1111/j.1742-4658.2008.06754.xISI: 000261184700025OAI: oai:DiVA.org:uu-86664DiVA: diva2:126842
Available from: 2008-11-27 Created: 2008-11-24 Last updated: 2010-08-18Bibliographically 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.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 707
Epoxide hydrolase; regioselectivity; selectivity; hysteresis; conformational changes; cooperativity; deep eutectic solvents; methylstyrene oxide
National Category
Biochemistry and Molecular Biology
Research subject
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)
Available from: 2010-01-28 Created: 2010-01-12 Last updated: 2010-01-28Bibliographically approved

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