Catalysis by theta-class glutathione transferases: Substrate binding, product formation and product release
2000 (English)Doctoral thesis, comprehensive summary (Other academic)
Glutathione transferases (GSTs) are detoxication enzymes that catalyze theconjugation of glutathione to a large number of electrophilic molecules. In this waythey help to rid cells of potentially toxic substances. This thesis deals with thecatalysis afforded by Theta-class glutathione transferases.
Systems for heterologous expression and purification of human GST T1-1 and ratGST T2-2 were developed. The enzymes were then subjected to kinetic analyses.
Both enzymes catalyze their reactions by lowering the pKa value for the thiol of glutathione, from 9.2 in solution to 5.7 in the enzymes' active sites. This is largely achieved by Ser11, which promotes stabilization but also formation of the reactive glutathione thiolate under physiological conditions.
In the reactions catalyzed by rat GST T2-2, product formation is fast, but product release from the enzyme is slow and rate limiting. Again Ser11 affects the kinetics; the S11A mutant has a 10-fold slower product release than the wild-type enzyme.
Conformational transitions in the enzyme's tertiary structure affect glutathione binding and catalysis. Possibly, GST T2-2 operates according to a hysteretic reaction mechanism where distinct enzyme conformations are assumed during steady-state catalysis, and at equilibrium, respectively.
The evolutionarily younger GST A1-1 of the Alpha class also displays kinetic effects related to conformational changes, but has evolved a faster glutathione binding due to a more accessible active site.
Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2000. , 56 p.
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 543
Biochemistry and Molecular Biology
Research subject Biochemistry
IdentifiersURN: urn:nbn:se:uu:diva-1061ISBN: 91-554-4735-XOAI: oai:DiVA.org:uu-1061DiVA: diva2:160601
2000-05-13, lecture hall B42, Biomedical Center, Uppsala, Uppsala, 10:15