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Residue 234 is a master switch of the alternative-substrate activity profile of human and rodent theta class glutathione transferase T1-1
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.
2010 (English)In: Biochimica et Biophysica Acta - General Subjects, ISSN 0304-4165, E-ISSN 1872-8006, Vol. 1800, no 4, 466-473 p.Article in journal (Refereed) Published
Abstract [en]

Background: The Theta class glutathione transferase GST T1-1 is a ubiquitously occurring detoxication enzyme. The rat and mouse enzymes have high catalytic activities with numerous electrophilic compounds, but the homologous human GST T1-1 has comparatively low activity with the same substrates. A major structural determinant of substrate recognition is the H-site, which binds the electrophile in proximity to the nucleophilic sulfur of the second substrate glutathione. The H-site is formed by several segments of amino acid residues located in separate regions of the primary structure. The C-terminal helix of the protein serves as a lid over the active site, and contributes several residues to the H-site. Methods: Site-directed mutagenesis of the H-site in GST T1-1 was used to create the mouse Arg234Trp for comparison with the human Trp234Arg mutant and the wild-type rat, mouse, and human enzymes. The kinetic properties were investigated with an array of alternative electrophilic substrates to establish substrate selectivity profiles for the different GST T1-1 variants. Results: The characteristic activity profile of the rat and mouse enzymes is dependent on Arg in position 234, whereas the human enzyme features Trp. Reciprocal mutations of residue 234 between the rodent and human enzymes transform the substrate-selectivity profiles from one to the other. Conclusions: H-site residue 234 has a key role in governing the activity and substrate selectivity profile of GST T1-1. General significance: The functional divergence between human and rodent Theta class GST demonstrates that a single point mutation can enable or suppress enzyme activities with different substrates.

Place, publisher, year, edition, pages
2010. Vol. 1800, no 4, 466-473 p.
Keyword [en]
Glutathione transferase, active site residue, alternative substrates, detoxication, haloalkanes
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-119506DOI: 10.1016/j.bbagen.2010.01.003ISI: 000275843100006PubMedID: 20097269OAI: oai:DiVA.org:uu-119506DiVA: diva2:300347
Available from: 2010-02-26 Created: 2010-02-26 Last updated: 2017-12-12Bibliographically approved
In thesis
1. Design of Glutathione Transferase Variants for Novel Activities with Alternative Substrates
Open this publication in new window or tab >>Design of Glutathione Transferase Variants for Novel Activities with Alternative Substrates
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Glutathione transferases (GSTs) play a pivotal role in cellular defense, since they are main contributors to the inactivation of genotoxic compounds of exogenous and endogenous origins. Directed evolution was used to improve the catalytic activities of Theta class GST T1-1 toward different substrates. The library was constructed by recombination of cDNA coding for human GST T1-1 and rodent Theta class GSTs, resulting in the F2-F5 generations. The clones were heterologously expressed in Escherichia coli and screened for variants with enhanced alkyltransferase activity. A mutant, F2:1215, with a 70-fold increased catalytic efficiency with 4-nitrophenethyl bromide (NPB) compared to human GST T1-1, was isolated from the second generation. NPB was used as a surrogate substrate of the anticancer drug 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in order to facilitate the screening process. The catalytic efficiency of the F2:1215 with BCNU had improved 170-fold compared to wild-type human GST T1-1, suggesting that NPB is a suitable model substrate for the anticancer drug BCNU. The sequence of the F2:1215 mutant differs from wild-type human GST T1-1 by three residues; one of these differences is Arg234, which corresponds to Trp in the human enzyme. Upon replacing the Trp234 in the human GST T1-1 with Arg, the resulting mutant (hTrp234Arg) showed enhanced alkyltransferase activity with a wide range of substrates (e.g. haloalkanes and other typical GSTs substrates). The three-dimensional structures of both wild-type human GST T1-1 and hTrp234Arg mutant help to explain the higher activity showed by of hTrp234Arg mutant compared to wild-type enzyme. The reciprocal mutation of the residue 234 in mouse GST T1-1 to that found in human, mArg234Trp, caused a dramatic decrease in the activity of the mouse enzyme to be similar to human GST T1-1. This indicates that residue 234 can be considered as a master switch of activities between human and rodent GST T1-1. Another important residue in the C-terminal helix of GST T1-1 is Met232. Although residue 232 points away from the H-site, it influences the catalytic activity and substrate selectivity of the mouse GST T1-1. A minor modification of Met232 induces major changes in the substrate-activity profile of the mouse GST T1-1 to favor novel substrates such as isothiocyanates and hydroperoxides and decreases the activity toward substrates that catalyzed by the wild-type enzyme.

 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2010. 77 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 720
National Category
Biochemistry and Molecular Biology Organic Chemistry
Research subject
Biochemistry; Organic Chemistry
Identifiers
urn:nbn:se:uu:diva-119768 (URN)978-91-554-7733-2 (ISBN)
Public defence
2010-04-12, C10:305, BMC, Husargatan 3, Uppsala, 10:15 (English)
Opponent
Supervisors
Available from: 2010-03-22 Created: 2010-03-01 Last updated: 2010-03-22Bibliographically approved

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