| dc.contributor.author |
Labrou, NE |
en |
| dc.contributor.author |
Mello, LV |
en |
| dc.contributor.author |
Clonis, YD |
en |
| dc.date.accessioned |
2014-06-06T06:44:44Z |
|
| dc.date.available |
2014-06-06T06:44:44Z |
|
| dc.date.issued |
2001 |
en |
| dc.identifier.issn |
00142956 |
en |
| dc.identifier.uri |
http://dx.doi.org/10.1046/j.1432-1327.2001.02307.x |
en |
| dc.identifier.uri |
http://62.217.125.90/xmlui/handle/123456789/2034 |
|
| dc.subject |
Cooperativity |
en |
| dc.subject |
Glutathione S-transferase |
en |
| dc.subject |
Herbicide detoxification |
en |
| dc.subject |
Protein engineering |
en |
| dc.subject.other |
1 chloro 2,4 dinitrobenzene |
en |
| dc.subject.other |
1 hydroxyl 2,4 dinitrobenzene |
en |
| dc.subject.other |
alanine |
en |
| dc.subject.other |
amino acid |
en |
| dc.subject.other |
asparagine |
en |
| dc.subject.other |
benzene derivative |
en |
| dc.subject.other |
enzyme |
en |
| dc.subject.other |
glutathione |
en |
| dc.subject.other |
glutathione transferase |
en |
| dc.subject.other |
monomer |
en |
| dc.subject.other |
phenylalanine |
en |
| dc.subject.other |
trypsin |
en |
| dc.subject.other |
unclassified drug |
en |
| dc.subject.other |
alpha helix |
en |
| dc.subject.other |
amino acid substitution |
en |
| dc.subject.other |
amino terminal sequence |
en |
| dc.subject.other |
article |
en |
| dc.subject.other |
binding site |
en |
| dc.subject.other |
catalysis |
en |
| dc.subject.other |
controlled study |
en |
| dc.subject.other |
enzyme substrate complex |
en |
| dc.subject.other |
enzyme subunit |
en |
| dc.subject.other |
hydrogen bond |
en |
| dc.subject.other |
kinetics |
en |
| dc.subject.other |
maize |
en |
| dc.subject.other |
nonhuman |
en |
| dc.subject.other |
priority journal |
en |
| dc.subject.other |
protein degradation |
en |
| dc.subject.other |
protein interaction |
en |
| dc.subject.other |
protein structure |
en |
| dc.subject.other |
site directed mutagenesis |
en |
| dc.subject.other |
thermostability |
en |
| dc.subject.other |
ultraviolet spectroscopy |
en |
| dc.subject.other |
viscosity |
en |
| dc.subject.other |
Alanine |
en |
| dc.subject.other |
Amino Acid Sequence |
en |
| dc.subject.other |
Asparagine |
en |
| dc.subject.other |
Catalysis |
en |
| dc.subject.other |
Conserved Sequence |
en |
| dc.subject.other |
Enzyme Stability |
en |
| dc.subject.other |
Glutathione Transferase |
en |
| dc.subject.other |
Heat |
en |
| dc.subject.other |
Herbicides |
en |
| dc.subject.other |
Kinetics |
en |
| dc.subject.other |
Models, Molecular |
en |
| dc.subject.other |
Mutagenesis, Site-Directed |
en |
| dc.subject.other |
Plant Proteins |
en |
| dc.subject.other |
Protein Subunits |
en |
| dc.subject.other |
Substrate Specificity |
en |
| dc.subject.other |
Trypsin |
en |
| dc.subject.other |
Viscosity |
en |
| dc.subject.other |
Zea mays |
en |
| dc.title |
The conserved Asn49 of maize glutathione S-transferase I modulates substrate binding, catalysis and intersubunit communication |
en |
| heal.type |
journalArticle |
en |
| heal.identifier.primary |
10.1046/j.1432-1327.2001.02307.x |
en |
| heal.publicationDate |
2001 |
en |
| heal.abstract |
The functional and structural role of the conserved Asn49 of theta class maize glutathione S-transferase was investigated by site-directed mutagenesis. Asn49 is located in the type I β turn formed by residues 49-52, and is involved in extensive hydrogen-bonding interactions between α helix 2 and the rest of the N-terminal domain. The substitution of Asn49 with Ala induces positive cooperativity for 1-chloro-2,4-dinitrobenzene (CDNB) binding as reflected by a Hill coefficient of 1.9 (S0.5CDNB = 0.43 mM). The positive cooperativity is also confirmed by following the isothermic binding of 1-hydroxyl-2,4-dinitrobenzene (HDNB) by UV-difference spectroscopy. In addition, the mutated enzyme exhibits: (a) an increase in the KmGSH value of about 6.5-fold, and decrease in kcat value of about fourfold; (b) viscosity-independent kinetic parameters; (c) lower thermostability, and (d) increased susceptibility to proteolytic attack by trypsin, when compared to the wild-type enzyme. It is concluded that Asn49 affects the rate-limiting step of the catalytic reaction, and contributes significantly to the structural and binding characteristics of both the glutathione binding site (G-site) and the electrophile substrate binding site (H-site) by affecting the structural integrity of a type I β turn (comprising residues 49-52) and probably the flexibility of the highly mobile short 310 helical segment of α helix 2 (residues 35-46). These structural perturbations are probably transmitted, via Phe51 and Phe65, to α helix H3″ of the adjacent subunit which contains key residues that interact with the electrophile substrate and contribute to the monomer-monomer contact region. This may accounts for the positive cooperativity observed. |
en |
| heal.journalName |
European Journal of Biochemistry |
en |
| dc.identifier.issue |
14 |
en |
| dc.identifier.volume |
268 |
en |
| dc.identifier.doi |
10.1046/j.1432-1327.2001.02307.x |
en |
| dc.identifier.spage |
3950 |
en |
| dc.identifier.epage |
3957 |
en |