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Human glutathione transferase T2-2 discloses some evolutionary strategies for optimization of substrate binding to the active site of glutathione transferases.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 2001 Feb 23; Vol. 276 (8), pp. 5427-31. Date of Electronic Publication: 2000 Oct 23. - Publication Year :
- 2001
-
Abstract
- Rapid kinetic, spectroscopic, and potentiometric studies have been performed on human Theta class glutathione transferase T2-2 to dissect the mechanism of interaction of this enzyme with its natural substrate GSH. Theta class glutathione transferases are considered to be older than Alpha, Pi, and Mu classes in the evolutionary pathway. As in the more recently evolved GSTs, the activation of GSH in the human Theta enzyme proceeds by a forced deprotonation of the sulfhydryl group (pK(a) = 6.1). The thiol proton is released quantitatively in solution, but above pH 6.5, a protein residue acts as an internal base. Unlike Alpha, Mu, and Pi class isoenzymes, the GSH-binding mechanism occurs via a simple bimolecular reaction with k(on) and k(off) values at least hundred times lower (k(on) = (2.7 +/- 0.8) x 10(4) M(-1) s(-1), k(off) = 36 +/- 9 s(-1), at 37 degrees C). Replacement of Arg-107 by alanine, using site-directed mutagenesis, remarkably increases the pK(a) value of the bound GSH and modifies the substrate binding modality. Y107A mutant enzyme displays a mechanism and rate constants for GSH binding approaching those of Alpha, Mu, and Pi isoenzymes. Comparison of available crystallographic data for all these GSTs reveals an unexpected evolutionary trend in terms of flexibility, which provides a basis for understanding our experimental results.
- Subjects :
- Arginine genetics
Flow Injection Analysis
Glutathione Transferase classification
Humans
Hydrogen-Ion Concentration
Isoenzymes genetics
Isoenzymes metabolism
Kinetics
Models, Chemical
Mutagenesis, Site-Directed
Mutation
Protons
Sulfhydryl Compounds metabolism
Catalytic Domain
Evolution, Molecular
Glutathione metabolism
Glutathione Transferase genetics
Glutathione Transferase metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 0021-9258
- Volume :
- 276
- Issue :
- 8
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 11044442
- Full Text :
- https://doi.org/10.1074/jbc.M002819200