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Implications for an Ionized Alkyl-Enzyme Intermediate during StEH1-Catalyzed trans-Stilbene Oxide Hydrolysis
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
2006 (English)In: Biochemistry, Vol. 45, 205-212 p.Article in journal (Refereed) Published
Abstract [en]

The catalytic mechanism of epoxide hydrolase (EC involves acid-assisted ring opening of the oxirane during the alkylation half-reaction of hydrolysis. Two tyrosyl residues in the active site of epoxide hydrolases have been shown to contribute to the catalysis of enzyme alkylation, but their mechanism of action has not been fully described. We have investigated the involvement of the active site Tyr154 and Tyr235 during S,S-trans-stilbene oxide hydrolysis catalyzed by potato epoxide hydrolase StEH1. Tyr phenol ionizations of unliganded enzyme as well as under pre-steady-state conditions during catalysis were studied by direct absorption spectroscopy. A transient UV absorption, indicative of tyrosinate formation, was detected during the lifetime of the alkyl-enzyme intermediate. The apparent pKa of Tyr ionization was 7.3, a value more than 3 pH units below the estimated pKa of protein Tyr residues in the unliganded enzyme. In addition, the pH dependencies of microscopic kinetic rates of catalyzed S,S-trans-stilbene oxide hydrolysis were determined. The alkylation rate increased with pH and displayed a pKa value identical to that of Tyr ionization (7.3), whereas the reverse (epoxidation) reaction did not display any pH dependence. The rate of alkyl-enzyme hydrolysis was inversely dependent on tyrosinate formation, decreasing with its buildup in the active site. Since alkyl-enzyme hydrolysis is the rate-limiting step of the overall reaction, kcat displayed the same decrease with pH as the hydrolysis rate. The compiled results suggested that the role of the Tyr154/Tyr235 pair was not as ultimate proton donor to the alkoxide anion but to stabilize the negatively charged alkyl-enzyme through electrophilic catalysis via hydrogen bonding.

Place, publisher, year, edition, pages
2006. Vol. 45, 205-212 p.
National Category
Biochemistry and Molecular Biology
Research subject
URN: urn:nbn:se:uu:diva-93348DOI: 10.1021/bi051893gOAI: oai:DiVA.org:uu-93348DiVA: diva2:166801
Available from: 2005-09-09 Created: 2005-09-09 Last updated: 2010-12-21
In thesis
1. Characterization of Epoxide Hydrolases from Yeast and Potato
Open this publication in new window or tab >>Characterization of Epoxide Hydrolases from Yeast and Potato
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Epoxides are three-membered cyclic ethers formed in the metabolism of foreign substances and as endogenous metabolites. Epoxide hydrolases (EHs) are enzymes that catalyze the hydrolysis of epoxides to yield the corresponding diols. EHs have been implicated in diverse functions such as detoxification of various toxic epoxides, as well as regulation of signal substance levels.

The main goal of this thesis was to investigate and characterize the α/β hydrolase fold EH. The first part concerns the identifictaion of an EH in Saccharomyces cerevisiae. The second part involves detailed mechanistic and structural studies of a plant EH from potato, StEH1.

Despite the important function of EH, no EH has previously been established in S. cerevisiae. By sequence analysis, we have identified a new subclass of EH present in yeast and in a wide range of microorganisms. The S. cerevisiae protein was produced recombinantly and was shown to display low catalytic activity with tested epoxide substrates.

In plants, EHs are involved in the general defence system, both in the metabolism of the cutin layer and in stress response to pathogens. The catalytic mechanism of recombinantly expressed wild type and mutant potato EH were investigated in detail using the two enantiomers of trans-stilbene oxide (TSO). The proposed catalytic residues of StEH1 were confirmed. StEH1 is slightly enantioselective for the S,S-enantiomer of trans-stilbene oxide. Furthermore, distinct pH dependence of the two enantiomers probably reflects differences in the microscopic rate constants of the substrates. The detailed function of the two catalytic tyrosines was also studied. The behavior of the tyrosine pair resembles that of a bidentate Lewis acid and we conclude that these tyrosines function as Lewis acids rather then proton donors.

The three dimensional structure of StEH1 was solved, representing the first structure of a plant EH. The structure provided information about the substrate specificity of StEH1.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2005. 50 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 80
Biochemistry, Epoxide hydrolase, Catalytic residues, Active site, trans-stilbene oxide, Rapid kinetics, Active site tyrosyls, Enzyme mechanism, Lewis acid, X-ray crystallography, Substrate specificity, Unidentified ORF, α/β hydrolase fold, Saccharomyces cerevisiae, Biokemi
National Category
Biochemistry and Molecular Biology
urn:nbn:se:uu:diva-5900 (URN)91-554-6315-0 (ISBN)
Public defence
2005-09-30, B22, BMC, Husarg. 5, Uppsala, 13:15
Available from: 2005-09-09 Created: 2005-09-09Bibliographically approved

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Widersten, Mikael
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