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The role of glutathione in the isomerization of Delta(5)-androstene-3,17-dione catalyzed by human glutathione transferase A1-1
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
2001 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 276, no 15, 11698-11704 p.Article in journal (Refereed) Published
Abstract [en]

Human glutathione transferase (GST) A1-1 efficiently catalyzes the isomerization of Delta(5)-androstene-3,17-dione (AD) into Delta(4)-androstene-3,17-dione. High activity requires glutathione, but enzymatic catalysis occurs also in the absence of this cofactor. Glutathione alone shows a limited catalytic effect. S-Alkylglutathione derivatives do not promote the reaction, and the pH dependence of the isomerization indicates that the glutathione thiolate serves as a base in the catalytic mechanism. Mutation of the active-site Tyr(9) into Phe significantly decreases the steady-state kinetic parameters, alters their pH dependence, and increases the pK(a) value of the enzyme-bound glutathione thiol. Thus, Tyr(9) promotes the reaction via its phenolic hydroxyl group in protonated form. GST A2-2 has a catalytic efficiency with AD 100-fold lower than the homologous GST A1-1. Another Alpha class enzyme, GST A4-4, is 1000-fold less active than GST A1-1. The Y9F mutant of GST A1-1 is more efficient than GST A2-2 and GST A4-4, both having a glutathione cofactor and an active-site Tyr(9) residue. The active sites of GST A2-2 and GST A1-1 differ by only four amino acid residues, suggesting that proper orientation of AD in relation to the thiolate of glutathione is crucial for high catalytic efficiency in the isomerization reaction. The GST A1-1-catalyzed steroid isomerization provides a complement to the previously described isomerase activity of 3beta-hydroxysteroid dehydrogenase.

Place, publisher, year, edition, pages
2001. Vol. 276, no 15, 11698-11704 p.
National Category
Natural Sciences
URN: urn:nbn:se:uu:diva-89851DOI: 10.1074/jbc.M009146200PubMedID: 11152686OAI: oai:DiVA.org:uu-89851DiVA: diva2:161670
Available from: 2002-05-03 Created: 2002-05-03 Last updated: 2013-06-13Bibliographically approved
In thesis
1. Alpha-class glutathione transferases as steroid isomerases and scaffolds for protein redesign
Open this publication in new window or tab >>Alpha-class glutathione transferases as steroid isomerases and scaffolds for protein redesign
2002 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The present work focuses on the glutathione transferase (GST) Alpha-class enzymes, their characteristics as steroid isomerases and structural plasticity as malleable scaffolds for protein design. The GSTs are a family of detoxication enzymes that appears to have a wider variety of additional functions.

Kinetic steady-state parameters for human GST A1-1 with the steroid isomerase substrate Δ5-androstene-3,17-dione (AD), an intermediate in steroid hormone biosynthesis, were determined. It was established that GST A1-1 is a highly efficient steroid isomerase with a 30-fold higher catalytic efficiency, in terms of kcat/Km, than 3β-hydroxysteroid dehydrogenase/Δ54-isomerase, the enzyme regarded as the mammalian Δ54-isomerase in steroid hormone biosynthesis. Kinetic parameters were also determined for GST A2-2, GST A4-4 and the GST A1-1 mutant Y9F. From the dependency on pH of the kinetic parameters it was established that efficient catalysis requires glutathione (GSH) in its deprotonated form and it is suggested that the GSH-thiolate acts as a base in the catalysis of the Δ54-3-ketosteroid isomerase reaction.

GST A2-2 is a poor catalyst of the steroid isomerase reaction while GST A3-3 is highly efficient. Their catalytic efficiencies (kcat/Km) differ 5000-fold. Stepwise point mutations were performed to GST A2-2 in order to insert the amino acid residues from the active-site of GST A3-3 that distinguishes the two isoenzymes. The result was that GST A2-2 was redesigned to a highly efficient double-bond isomerase with both the catalytic constant (kcat) and catalytic efficiency (kcat/Km) in the same order as for GST A3-3. Furthermore, this was done by only exchanging amino-acid residues with first-sphere interactions, providing empirical proof-of principle for knowledge-based enzyme design.

Kinetic studies on GST A1-1 and a T68E mutant of GST A1-1 were also performed with a GSH analog lacking the g-glutamate a-carboxylate (dGSH), and using three different electrophilic substrates (AD; 1-chloro-2,4-dinitrobenzene, CDNB; 4-nitrocinnamaldehyde). Deletion of the a-carboxylate from the GSH glutamate had a severe impact on all reaction constants and it changed the rate-limiting step for the CDNB reaction as well as changed the pKa value for the enzyme-bound GSH thiol. The loss in activity caused by dGSH could in part be compensated by the T68E mutant contributing an enzyme-bound carboxylate instead.

The C-terminus of GST A1-1 is flexible and folds over the active site when the enzyme binds a substrate. Phenylalanine residues in the C-terminal end, known to interact with active-site residues tyrosine 9 and phenylalanine 10, were mutated to abolish those interactions. Studies of viscosity dependence for CDNB and AD with regard to kcat and kcat/Km showed that the dynamic C-terminal segment influence rate-determining steps for both the larger isomerase substrate, AD, as well as for the smaller conjugation substrate, CDNB.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2002. 38 p.
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 719
Biochemistry, Biokemi
National Category
Biochemistry and Molecular Biology
Research subject
urn:nbn:se:uu:diva-2034 (URN)91-554-5327-9 (ISBN)
Public defence
2002-05-28, BMC B22, Uppsala, 10:15
Available from: 2002-05-03 Created: 2002-05-03 Last updated: 2014-01-27Bibliographically approved

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