| dc.contributor.author |
Kotzia, GA |
en |
| dc.contributor.author |
Labrou, NE |
en |
| dc.date.accessioned |
2014-06-06T06:46:06Z |
|
| dc.date.available |
2014-06-06T06:46:06Z |
|
| dc.date.issued |
2004 |
en |
| dc.identifier.issn |
00142956 |
en |
| dc.identifier.uri |
http://dx.doi.org/10.1111/j.0014-2956.2004.04285.x |
en |
| dc.identifier.uri |
http://62.217.125.90/xmlui/handle/123456789/2794 |
|
| dc.subject |
Affinity labelling |
en |
| dc.subject |
Chlorotriazine |
en |
| dc.subject |
Herbicides |
en |
| dc.subject |
Xenobiotics |
en |
| dc.subject.other |
amino acid |
en |
| dc.subject.other |
glutathione derivative |
en |
| dc.subject.other |
glutathione transferase |
en |
| dc.subject.other |
isoenzyme |
en |
| dc.subject.other |
reagent |
en |
| dc.subject.other |
s (2,3 dichlorotriazinyl)glutathione |
en |
| dc.subject.other |
unclassified drug |
en |
| dc.subject.other |
xenobiotic agent |
en |
| dc.subject.other |
affinity labeling |
en |
| dc.subject.other |
alkylation |
en |
| dc.subject.other |
alpha helix |
en |
| dc.subject.other |
amino acid analysis |
en |
| dc.subject.other |
article |
en |
| dc.subject.other |
concentration response |
en |
| dc.subject.other |
controlled study |
en |
| dc.subject.other |
enzyme active site |
en |
| dc.subject.other |
enzyme binding |
en |
| dc.subject.other |
enzyme inactivation |
en |
| dc.subject.other |
enzyme kinetics |
en |
| dc.subject.other |
enzyme modification |
en |
| dc.subject.other |
enzyme specificity |
en |
| dc.subject.other |
enzyme subunit |
en |
| dc.subject.other |
hydrophobicity |
en |
| dc.subject.other |
maize |
en |
| dc.subject.other |
molecular model |
en |
| dc.subject.other |
molecular probe |
en |
| dc.subject.other |
nonhuman |
en |
| dc.subject.other |
priority journal |
en |
| dc.subject.other |
protein motif |
en |
| dc.subject.other |
site directed mutagenesis |
en |
| dc.subject.other |
structure activity relation |
en |
| dc.subject.other |
Affinity Labels |
en |
| dc.subject.other |
Animals |
en |
| dc.subject.other |
Binding Sites |
en |
| dc.subject.other |
Enzyme Inhibitors |
en |
| dc.subject.other |
Glutathione |
en |
| dc.subject.other |
Glutathione Transferase |
en |
| dc.subject.other |
Models, Molecular |
en |
| dc.subject.other |
Molecular Structure |
en |
| dc.subject.other |
Mutagenesis, Site-Directed |
en |
| dc.subject.other |
Plant Proteins |
en |
| dc.subject.other |
Protein Binding |
en |
| dc.subject.other |
Protein Conformation |
en |
| dc.subject.other |
Protein Subunits |
en |
| dc.subject.other |
Zea mays |
en |
| dc.subject.other |
Zea mays |
en |
| dc.title |
S-(2,3-dichlorotriazinyl)glutathione: A new affinity label for probing the structure and function of glutathione transferases |
en |
| heal.type |
journalArticle |
en |
| heal.identifier.primary |
10.1111/j.0014-2956.2004.04285.x |
en |
| heal.publicationDate |
2004 |
en |
| heal.abstract |
S-(2,3-Dichlorotriazinyl)glutathione (SDTG) was synthesized and shown to be an effective alkylating affinity label for recombinant maize glutathione S-transferase I (GST I). Inactivation of GST I by SDTG at pH 6.5 followed biphasic pseudo-first-order saturation kinetics. The biphasic kinetics can be described in terms of a fast initial phase of inactivation followed by a slower phase, leading to 42 ± 3% residual activity. The rate of inactivation for both phases exhibits nonlinear dependence on SDTG concentration, consistent with the formation of a reversible complex with the enzyme (Kd 107.9 ± 2.1 μM for the fast phase, and 224.5 ± 4.2 μM for the slow phase) before irreversible modification with maximum rate constants of 0.049 ± 0.002 min-1 and 0.0153 ± 0.001 min-1 for the fast and slow phases, respectively. Protection from inactivation was afforded by substrate analogues, demonstrating the specificity of the reaction. When the enzyme was inactivated (42% residual activity), ≈ 1 mol SDTG per mol dimeric enzyme was incorporated. Amino-acid analysis, molecular modelling, and site-directed mutagenesis studies suggested that the modifying residue is Met121, which is located at the end of α-helix H‴3 and forms part of the xenobiotic-binding site. The results reveal an unexpected structural communication between subunits, which consists of mutually exclusive modification of Met residues across enzyme subunits. Thus, modification of Met121 on one subunit prevents modification of Met121 on the other subunit. This communication is governed by Phe51, which is located at the dimer interface and forms part of the hydrophobic lock-and-key intersubunit motif. The ability of SDTG to inactivate other glutathione-binding enzymes and GST isoenzymes was also investigated, and it was concluded that this new reagent may have general applicability as an affinity reagent for other enzymes with glutathione-binding sites. |
en |
| heal.journalName |
European Journal of Biochemistry |
en |
| dc.identifier.issue |
17 |
en |
| dc.identifier.volume |
271 |
en |
| dc.identifier.doi |
10.1111/j.0014-2956.2004.04285.x |
en |
| dc.identifier.spage |
3503 |
en |
| dc.identifier.epage |
3511 |
en |