Logo: to the web site of Uppsala University

uu.sePublications from Uppsala University
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Exploring Selectivity and Hysteresis: Kinetic Studies on a Potato Epoxide Hydrolase
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry. (Mikael Widersten)
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. , p. 62
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 707
Keywords [en]
Epoxide hydrolase; regioselectivity; selectivity; hysteresis; conformational changes; cooperativity; deep eutectic solvents; methylstyrene oxide
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:uu:diva-112285ISBN: 978-91-554-7701-1 (print)OAI: oai:DiVA.org:uu-112285DiVA, id: diva2:285671
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
List of papers
1. Substrate-dependent hysteretic behavior in StEH1-catalyzed hydrolysis of styrene oxide derivatives
Open this publication in new window or tab >>Substrate-dependent hysteretic behavior in StEH1-catalyzed hydrolysis of styrene oxide derivatives
2008 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 275, no 24, p. 6309-6320Article 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.

National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-86664 (URN)10.1111/j.1742-4658.2008.06754.x (DOI)000261184700025 ()
Available from: 2008-11-27 Created: 2008-11-24 Last updated: 2017-12-14Bibliographically approved
2. Mutations in salt-bridging residues at the interface of the core and lid domains of epoxide hydrolase StEH1 affect regioselectivity, protein stability and hysteresis
Open this publication in new window or tab >>Mutations in salt-bridging residues at the interface of the core and lid domains of epoxide hydrolase StEH1 affect regioselectivity, protein stability and hysteresis
2010 (English)In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 495, no 2, p. 165-173Article 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
National Category
Natural Sciences Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-112278 (URN)10.1016/j.abb.2010.01.007 (DOI)000275299800009 ()20079707 (PubMedID)
Available from: 2010-01-12 Created: 2010-01-12 Last updated: 2017-12-12Bibliographically approved
3. Temperature and pH dependence of enzyme catalyzed hydrolysis of trans-methylstyrene oxide: a unifying kinetic model for observed hysteresis, cooperativity and regioselectivity
Open this publication in new window or tab >>Temperature and pH dependence of enzyme catalyzed hydrolysis of trans-methylstyrene oxide: a unifying kinetic model for observed hysteresis, cooperativity and regioselectivity
2010 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 49, no 10, p. 2297-2304Article 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.

Keywords
epoxide hydrolase, regioselectivity, hysteresis, StEH1
National Category
Chemical Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-112272 (URN)10.1021/bi902157b (DOI)000275227000022 ()20146441 (PubMedID)
Available from: 2010-01-12 Created: 2010-01-12 Last updated: 2017-12-12Bibliographically approved
4. Deep eutectic solvents (DESs) are viable cosolvents for enzyme-catalyzed epoxide hydrolysis
Open this publication in new window or tab >>Deep eutectic solvents (DESs) are viable cosolvents for enzyme-catalyzed epoxide hydrolysis
2010 (English)In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 147, no 3-4, p. 169-171Article in journal (Refereed) Published
Abstract [en]

A special group of ionic liquids, deep eutectic solvents (DESs) have been tested as cosolvents in enzyme-catalyzed hydrolysis of a chiral (1,2)-trans-2-methylstyrene oxide. The choline chloride:ethane diol (ET), choline chloride:glycerol (GLY) and choline:chloride:urea (REL) DESs were included in the reaction mixtures with epoxide and the potato epoxide hydrolase StEH1. The effect of the DESs on enzyme function was primarily elevations of KM (up to 20-fold) and with lesser effects on turnover numbers (two-fold variation). The regioselectivity in hydrolysis of the (1R,2R)-2-trans-methylstyrene oxide was altered in the presence of GLY or ET to favor epoxide ring opening at the benzylic carbon (R=2.33), enhancing the regioselectivity observed in buffer-only systems (R=1.35). The DES solutions dissolved 1.5-fold higher epoxide concentrations as compared to phosphate buffer. The total conversion of high concentration (40 g/l) of (1S,2S)-MeSO was not negatively affected by addition of 40 % GLY.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-112276 (URN)10.1016/j.jbiotec.2010.04.011 (DOI)000279408600005 ()
Available from: 2010-01-12 Created: 2010-01-12 Last updated: 2017-12-12Bibliographically approved

Open Access in DiVA

fulltext(2258 kB)878 downloads
File information
File name FULLTEXT01.pdfFile size 2258 kBChecksum SHA-512
acc24371852cce97cc7cdc3be49d556dfa83fa84f53b7f63e8a51e56a4aa11a2b8032b94ff254bb9310e7f0e76c11e6114e4155c6100f93851d6ccb604ec0aa3
Type fulltextMimetype application/pdf

By organisation
Department of Biochemistry and Organic Chemistry
Biochemistry and Molecular Biology

Search outside of DiVA

GoogleGoogle Scholar
Total: 878 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 894 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf