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Mutations in salt-bridging residues at the interface of the core and lid domains of epoxide hydrolase StEH1 affect regioselectivity, protein stability and hysteresis
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry. (Widersten)
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. (Mikael Widersten)
2010 (English)In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 495, no 2, 165-173 p.Article in journal (Refereed) Published
Abstract [en]

Epoxide hydrolase, StEH1, shows hysteretic behavior in the catalyzed hydrolysis of trans-2-methylstyrene oxide (2-MeSO)(1). Linkage between protein structure dynamics and catalytic function was probed in mutant enzymes in which surface-located salt-bridging residues were substituted. Salt-bridges at the interface of the alpha/beta-hydrolase fold core and lid domains, as well as between residues in the lid domain, between Lys(179)Asp(202), Glu(215)-Arg(41) and Arg(236)-Glu(136) were disrupted by mutations, K179Q E215Q, R236Q and R236Q. All mutants displayed enzyme activity with styrene oxide (SO) and 2-MeSO when assayed at 30 degrees C. Disruption of salt-bridges altered the rates for isomerization between distinct Michaelis complexes, with (1R,2R)-2-MeSO as substrate, presumably as a result of increased dynamics of involved protein segments. Another indication of increased flexibility was a lowered thermostability in all mutants. We propose that the alterations to regioselectivity in these mutants derive from an increased mobility in protein segments otherwise stabilized by salt bridging interactions.

Place, publisher, year, edition, pages
Amsterdam: Elsevier , 2010. Vol. 495, no 2, 165-173 p.
National Category
Natural Sciences Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:uu:diva-112278DOI: 10.1016/j.abb.2010.01.007ISI: 000275299800009PubMedID: 20079707OAI: oai:DiVA.org:uu-112278DiVA: diva2:285640
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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