Engineering the pH-dependence of kinetic parameters of maize glutathione S-transferase I by site-directed mutagenesis

dc.contributor.author	Labrou, NE	en
dc.contributor.author	Rigden, DJ	en
dc.contributor.author	Clonis, YD	en
dc.date.accessioned	2014-06-06T06:45:58Z
dc.date.available	2014-06-06T06:45:58Z
dc.date.issued	2004	en
dc.identifier.issn	13890344	en
dc.identifier.uri	http://dx.doi.org/10.1016/j.bioeng.2003.10.002	en
dc.identifier.uri	http://62.217.125.90/xmlui/handle/123456789/2741
dc.subject	1-chloro-2,4-dinitrobenzene	en
dc.subject	Catalytic mechanism	en
dc.subject	CDNB	en
dc.subject	Directed mutagenesis	en
dc.subject	Glutathione	en
dc.subject	Glutathione S-transferase	en
dc.subject	GSH	en
dc.subject	GST	en
dc.subject	Herbicide detoxification	en
dc.subject	pH-profile	en
dc.subject	Protein engineering	en
dc.subject.other	asparagine	en
dc.subject.other	glutamine	en
dc.subject.other	glutathione transferase	en
dc.subject.other	histidine	en
dc.subject.other	lysine	en
dc.subject.other	protein	en
dc.subject.other	serine	en
dc.subject.other	article	en
dc.subject.other	binding site	en
dc.subject.other	catalysis	en
dc.subject.other	chemical reaction kinetics	en
dc.subject.other	conformation	en
dc.subject.other	enzyme binding	en
dc.subject.other	enzyme engineering	en
dc.subject.other	enzyme mechanism	en
dc.subject.other	ionization	en
dc.subject.other	kinetics	en
dc.subject.other	nonhuman	en
dc.subject.other	pH measurement	en
dc.subject.other	phase transition	en
dc.subject.other	priority journal	en
dc.subject.other	site directed mutagenesis	en
dc.subject.other	thermodynamics	en
dc.subject.other	ultraviolet spectrophotometry	en
dc.subject.other	wild type	en
dc.subject.other	Amino Acid Substitution	en
dc.subject.other	Binding Sites	en
dc.subject.other	Glutathione Transferase	en
dc.subject.other	Hydrogen-Ion Concentration	en
dc.subject.other	Kinetics	en
dc.subject.other	Mutagenesis, Site-Directed	en
dc.subject.other	Plant Proteins	en
dc.subject.other	Protein Engineering	en
dc.subject.other	Substrate Specificity	en
dc.subject.other	Zea mays	en
dc.subject.other	Zea mays	en
dc.title	Engineering the pH-dependence of kinetic parameters of maize glutathione S-transferase I by site-directed mutagenesis	en
heal.type	journalArticle	en
heal.identifier.primary	10.1016/j.bioeng.2003.10.002	en
heal.publicationDate	2004	en
heal.abstract	The optimisation of enzymes for particular application or conditions remains an important target in all protein engineering endeavours. Here, we report a successful strategy for altering the pH-profile of kinetic parameters and to define in detail the molecular mechanism of maize glutathione S-transferase I (GST I). To accomplish this, selected residues from the glutathione binding site (His40, Ser11, Lys41, Asn49, Gln53 and Ser67) were mutated to Ala, and the pH-dependence of the catalytic parameters V max, and Vmax/KmGSH of the mutated forms were analysed. The pH-dependence of Vmax for the wild-type enzyme exhibits two transitions in the acidic pH range with pKa1 of 5.7 and pKa2 of 6.6. Based on thermodynamic data, site-directed mutagenesis and UV deference spectroscopy, it was concluded that pKa1 corresponds to GSH carboxylates, whereas the pKa2 has a conformational origin of the protein. The pH-dependence of Vmax/ KmGSH for the wild-type enzyme exhibits a single transition with pKa of 6.28 which was attributed to the thiol ionisation of bound GSH. These findings complement the conclusions about the catalytic mechanism deduced from the crystal structure of the enzyme and provide the basis for rationally designing engineered forms of GST I with valuable properties. © 2003 Elsevier B.V. All rights reserved.	en
heal.journalName	Biomolecular Engineering	en
dc.identifier.issue	2	en
dc.identifier.volume	21	en
dc.identifier.doi	10.1016/j.bioeng.2003.10.002	en
dc.identifier.spage	61	en
dc.identifier.epage	66	en