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Probing the Alpha Class Glutathione Transferase Structure. Combinatorial Subunit Studies and Rational Redesign
Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry.
2001 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The present work concerns two Alpha class enzymes, GST A1-1 and GST A4-4, of the glutathione transferase (GST) family, which are detoxication enzymes catalyzing the conjugation of the tripeptide glutathione with various electrophiles.

The GST A1-1 structure was explored for protein engineering purposes and tailored for increased activity toward alkenals. The target residues were rationally chosen and were mainly located within the first-sphere of coordination from the electrophilic substrate. The catalytic efficiency was increased 300-fold, resulting in an activity 3-fold greater than that of wild-type GST A4-4, an enzyme naturally evolved for alkenal activity.

Furthermore, half-of-the-sites reactivity was for the first time demonstrated in a GST. A heterodimer, composed of an inactive D101K-mutant subunit and a wild-type subunit, displayed catalytic activity identical to that of the wild-type homodimer in the Michael addition of glutathione to nonenal. However, both subunits in the wild-type dimer were active in the aromatic substitution reaction. The involvement of both subunits in catalysis was further demonstrated in a study of a heterodimeric GST A1-1 M208K-M208E variant, where the M208K mutant could be activated by benzoic acid derivatives.

The production of a GST A1-4 heterodimer proved that the two, rather diverse, isoenzymes are capable of hybridization, but the yield was limited. The heterodimer displayed a reasonable activity toward three different electrophiles. It was therefore proposed that two Alpha class specific dimer interfaces have evolved to restrain cross-hybridization between the A4 subunit and other isoenzymes of the Alpha class.

A kinetic investigation of GST A1-1 showed that catalysis was limited by product release in the CDNB reaction. Deletion of the α-carboxylate from the γ-Glu part of glutathione changed the rate-limiting step and raised the pKa value of the active-site-bound thiol. Furthermore, the α-carboxylate proved to be important for binding of both glutathione and the electrophilic substrate. The lack of the α-carboxylate could partially be compensated by the introduction of a glutamate containing a carboxylate in position 68 of the enzyme.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2001. , 55 p.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 609
Keyword [en]
Biochemistry, Glutathione transferases, rational redesign, enzyme, catalysis
Keyword [sv]
Biokemi
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biotechnology
Identifiers
URN: urn:nbn:se:uu:diva-1261ISBN: 91-554-4949-2 (print)OAI: oai:DiVA.org:uu-1261DiVA: diva2:160823
Public defence
2001-03-16, Sal B21, Uppsala Biomedical Centre, Husargatan 3, 751 23 Uppsala, Uppsala, 10:15
Available from: 2001-02-23 Created: 2001-02-23Bibliographically approved

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