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Structure-activity relationships and thermal stability of human glutathione transferase P1-1 governed by the H-site residue 105
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
1998 (English)In: Journal of Molecular Cell Biology, ISSN 1674-2788, Vol. 278, no 3, 687-698 p.Article in journal (Refereed) Published
Abstract [en]

Human glutathione transferase P1-1 (GSTP1-1) is polymorphic in amino acid residue 105, positioned in the substrate binding H-site. To elucidate the role of this residue an extensive characterization of GSTP1-1/Ile105 and GSTP1-1/Val105 was performed. Mutant enzymes with altered volume and hydrophobicity of residue 105, GSTP1-1/Ala105 and GSTP1-1/Trp105, were constructed and included in the study. Steady-state kinetic parameters and specific activities were determined using a panel of electrophilic substrates, with the aim of covering different types of reaction mechanisms. Analysis of the steady-state kinetic parameters indicates that the effect of the substitution of the amino acid in position 105 is highly dependent on substrate used. When 1-chloro-2,4-dinitrobenzene was used as substrate a change in the side-chain of residue 105 seemed primarily to cause changes in the KM value, while the kcat value was not distinctively affected. With other substrates, such as 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and ethacrynic acid both kcat and KM values were altered by the substitution of amino acid 105. The constant for formation of the sigma-complex between 1,3, 5-trinitrobenzene and glutathione was shown to be dependent upon the volume of the amino acid in position 105. The nature of the amino acid in position 105 was also shown to affect the thermal stability of the enzyme at 50 degrees C, indicating an important role for this residue in the stabilization of the enzyme. The GSTP1-1/Ile105 variant was approximately two to three times more stable than the Val105 variant as judged by their half-lives. The presence of glutathione in the incubation buffer afforded a threefold increase in the half-lives of the enzymes. Thus, the thermal stability of the enzyme and depending on substrate, both KM values and turnover numbers are influenced by substitutions in position 105 of GSTP1-1.

Place, publisher, year, edition, pages
1998. Vol. 278, no 3, 687-698 p.
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:uu:diva-89714DOI: 10.1006/jmbi.1998.1708PubMedID: 9600848OAI: oai:DiVA.org:uu-89714DiVA: diva2:161422
Available from: 2002-03-27 Created: 2002-03-27 Last updated: 2013-05-30Bibliographically approved
In thesis
1. Exploring the Functional Plasticity of Human Glutathione Transferases: Allelic Variants, Novel Isoenzyme and Enzyme Redesign
Open this publication in new window or tab >>Exploring the Functional Plasticity of Human Glutathione Transferases: Allelic Variants, Novel Isoenzyme and Enzyme Redesign
2002 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Glutathione transferases (GSTs) make up a superfamily that is involved in the cellular defense against various reactive compounds by catalyzing the conjugation of glutathione to electrophilic centra. Members of this family have also been implicated in different facets of biological signaling.

The gene encoding human GST P1-1 is polymorphic, resulting in variant amino acid residues in positions 105 and 114. The role of the polymorphism in the active-site residue 105 on enzyme stability and activity with various substrates was investigated. A valine instead of an isoleucine in position 105 decreased the thermal stability of the enzyme. The effect on enzyme activity was dependent on the substrate and reaction studied. With some substrates tested, such as carcinogenic diolepoxides derived from polyaromatic hydrocarbons, GST P1-1/Val105 displayed the highest catalytic efficiency. In contrast, with 1-chloro-2,4-dinitrobenzene, the GST P1-1/Ile105 showed higher activity. Residue 105 was mutated to alanine and tryptophan to investigate the role of size and hydrophobicity of residue 105 on enzyme properties. Generally, a smaller amino acid in position 105 gave increased activity with large substrates. Clearly, residue 105 of GST P1-1 helps to determine the substrate selectivity of the enzyme. In addition, more voluminous amino acids in position 105 increase the thermal stability of the enzyme.

GST P1-1 is believed to contribute to the development of drug resistance in cancer cells. The affinity of GST P1-1 for TER 117, designed to inhibit GST P1-1 in tumors, was not affected by the variability in position 105. TER 117 was found to be a potent inhibitor of glyoxalase I as well.

The cDNA encoding GST A3-3 was isolated from a placental cDNA library. GST A3-3 was heterologously expressed, purified and found to catalyze efficiently the double-bond isomerization of Δ5-androstene-3,17-dione and Δ5-pregnene-3,20-dione, reactions taking place in the biosynthesis of the steroid hormones testosterone and progesterone, respectively. GST A3-3 was found to be selectively expressed in steroidogenic tissues, suggesting that this enzyme is involved in the production of steroid hormones. The presence of both the hydroxyl group of the active-site tyrosine 9 and the thiolate form of glutathione, acting as a cofactor, is important for high double-bond isomerase activity. A leucine in position 111 appears to have a major role in productive binding of the steroid substrate but also residues F10 and A216 are determinants for the high isomerase activity.

GST A2-2 is a poor catalyst of the steroid double-bond isomerization of Δ5-androstene-3,17-dione as compared to GST A3-3, despite 88% sequence identity. GST A2-2 was redesigned to a highly efficient double-bond isomerase by mutating five active-site residues to the corresponding residues of GST A3-3. This demonstrates the functional plasticity of GSTs and the power of a rational approach to redesign of these enzymes.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2002. 56 p.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 695
Keyword
Biochemistry, Biokemi
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-1858 (URN)91-554-5270-1 (ISBN)
Public defence
2002-04-19, B42, Biomedical Center, Uppsala, 10:15
Opponent
Available from: 2002-03-27 Created: 2002-03-27Bibliographically approved

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Widersten, MikaelMannervik, Bengt

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