Functional and structural roles of the glutathione-binding residues in maize (Zea mays) glutathione S-transferase

dc.contributor.author	Labrou, NE	en
dc.contributor.author	Mello, LV	en
dc.contributor.author	Clonis, YD	en
dc.date.accessioned	2014-06-06T06:44:39Z
dc.date.available	2014-06-06T06:44:39Z
dc.date.issued	2001	en
dc.identifier.issn	02646021	en
dc.identifier.uri	http://dx.doi.org/10.1042/0264-6021:3580101	en
dc.identifier.uri	http://62.217.125.90/xmlui/handle/123456789/1996
dc.subject	Essential dynamics	en
dc.subject	Herbicide detoxification	en
dc.subject	Molecular dynamics	en
dc.subject	Protein engineering	en
dc.subject.other	Crops	en
dc.subject.other	Escherichia coli	en
dc.subject.other	Ionization	en
dc.subject.other	Mutagenesis	en
dc.subject.other	Spectroscopy	en
dc.subject.other	Enzyme catalytic mechanisms	en
dc.subject.other	Enzymes	en
dc.subject.other	1 chloro 2,4 dinitrobenzene	en
dc.subject.other	glutamine	en
dc.subject.other	glutathione transferase	en
dc.subject.other	histidine	en
dc.subject.other	lactoylglutathione lyase	en
dc.subject.other	lysine	en
dc.subject.other	phenylalanine	en
dc.subject.other	serine	en
dc.subject.other	alpha helix	en
dc.subject.other	article	en
dc.subject.other	binding site	en
dc.subject.other	catalysis	en
dc.subject.other	computer simulation	en
dc.subject.other	controlled study	en
dc.subject.other	enzyme analysis	en
dc.subject.other	enzyme kinetics	en
dc.subject.other	enzyme structure	en
dc.subject.other	Escherichia coli	en
dc.subject.other	maize	en
dc.subject.other	molecular cloning	en
dc.subject.other	molecular interaction	en
dc.subject.other	nonhuman	en
dc.subject.other	priority journal	en
dc.subject.other	protein degradation	en
dc.subject.other	protein expression	en
dc.subject.other	protein stability	en
dc.subject.other	site directed mutagenesis	en
dc.subject.other	thermodynamics	en
dc.subject.other	Binding Sites	en
dc.subject.other	Cloning, Molecular	en
dc.subject.other	Computer Simulation	en
dc.subject.other	Escherichia coli	en
dc.subject.other	Glutathione	en
dc.subject.other	Glutathione Transferase	en
dc.subject.other	Hydrogen-Ion Concentration	en
dc.subject.other	Kinetics	en
dc.subject.other	Models, Chemical	en
dc.subject.other	Models, Molecular	en
dc.subject.other	Mutagenesis, Site-Directed	en
dc.subject.other	Point Mutation	en
dc.subject.other	Protein Binding	en
dc.subject.other	Spectrophotometry	en
dc.subject.other	Sulfhydryl Compounds	en
dc.subject.other	Trypsin	en
dc.subject.other	Zea mays	en
dc.title	Functional and structural roles of the glutathione-binding residues in maize (Zea mays) glutathione S-transferase	en
heal.type	journalArticle	en
heal.identifier.primary	10.1042/0264-6021:3580101	en
heal.publicationDate	2001	en
heal.abstract	The isoenzyme glutathione S-transferase (GST) I from maize (Zea mays) was cloned and expressed in Escherichia coli, and its catalytic mechanism was investigated by site-directed mutagenesis and dynamic studies. The results showed that the enzyme promotes proton dissociation from the GSH thiol and creates a thiolate anion with high nucleophilic reactivity by lowering the pKa of the thiol from 8.7 to 6.2. Steady-state kinetics fit well to a rapid equilibrium, random sequential Bi Bi mechanism, with intrasubunit modulation between the GSH binding site (G-site) and the electrophile binding site (H-site). The rate-limiting step of the reaction is viscosity-dependent, and thermodynamic data suggest that product release is rate-limiting. Five residues of GST I (Ser11, His40, Lys41, Gln53 and Ser67), which are located in the G-site, were individually replaced with alanine and their structural and functional roles in the 1-chloro-2,4-dinitrobenzene (CDNB) conjugation reaction were investigated. On the basis of steady-state kinetics, difference spectroscopy and limited proteolysis studies it is concluded that these residues: (1) contribute to the affinity of the G-site for GSH, as they are involved in side-chain interaction with GSH; (2) influence GSH thiol ionization, and thus its reactivity; (3) participate in kcat regulation by affecting the rate-limiting step of the reaction; and (4) in the cases of His40, Lys41 and Gin53 play an important role in the structural integrity of, and probably in the flexibility of, the highly mobile short 310-helical segment of α-helix 2 (residues 35-46), as shown by limited proteolysis experiments. These structural perturbations are probably transmitted to the H-site through changes in Phe35 conformation. This accounts for the modulation of KmCDNB by His40, Lys41 and Gln53, and also for the intrasubunit communication between the G- and H-sites. Computer simulations using CONCOORD were applied to maize GST I monomer and dimer structures, each with bound lactoylglutathione, and the results were analysed by the essential dynamics technique. Differences in dynamics were found between the monomer and the dimer simulations showing the importance of using the whole structure in dynamic analysis. The results obtained confirm that the short 310-helical segment of α-helix 2 (residues 35-46) undergoes the most significant structural rearrangements. These rearrangements are discussed in terms of enzyme catalytic mechanism.	en
heal.journalName	Biochemical Journal	en
dc.identifier.issue	1	en
dc.identifier.volume	358	en
dc.identifier.doi	10.1042/0264-6021:3580101	en
dc.identifier.spage	101	en
dc.identifier.epage	110	en