title: Enzyme kinetics and substrate selectivities of rat glutathione S-transferase isoenzymes towards a series of new 2-substituted 1-chloro-4-nitrobenzenes.
abstract: 1. Four different rat glutathione S-transferase (GST) isoenzymes, belonging to three different classes, were examined for their GSH conjugating capacity towards 11 2-substituted 1-chloro-4-nitrobenzene derivatives. Significant differences were found in their enzyme kinetic parameters Km, kcat and kcat/Km. 2. Substrates with bulky substituents on the ortho-position appeared to have high affinities (low Km's) for the active site of the GST-isoenzymes, suggesting that there is sufficient space in this area of the active site. A remarkably high Km (low affinity) was found for 2-chloro-5-nitropyridine towards all GST-isoenzymes examined. 3. GST 3-3 catalysed the reaction between GSH and the substrates most efficiently (high kcat) compared with the other GST-isoenzymes. Moreover, GST 3-3 showed clear substrate selectivities towards the substrates with a trifluoromethyl-, chlorine- and bromine-substituent. 1-Chloro-2,4-dinitrobenzene and 2-chloro-5-nitrobenzonitrile were most efficiently conjugated by all four GST-isoenzymes examined. 4. When the rate of the conjugation reactions was followed, a linear increase of formation of GS-conjugate could be seen for 2-chloro-5-nitrobenzonitrile during a much longer period of time than for 1-chloro-2,4-dinitrobenzene with all GST-isoenzymes examined. Therefore, it is suggested that 2-chloro-5-nitrobenzonitrile might be recommended as an alternative model substrate in GST-research.
authors.affiliation: Department of Pharmacochemistry, Vrije Universiteit, Amsterdam, Netherlands.
authors.forename: E M
authors.initials: EM
authors.lastname: van der Aar
authors.forename: D
authors.initials: D
authors.lastname: Buikema
authors.forename: J N
authors.initials: JN
authors.lastname: Commandeur
authors.forename: J M
authors.initials: JM
authors.lastname: te Koppele
authors.forename: B
authors.initials: B
authors.lastname: van Ommen
authors.forename: P J
authors.initials: PJ
authors.lastname: van Bladeren
authors.forename: N P
authors.initials: NP
authors.lastname: Vermeulen
mesh_terms: D000818:Animals; D002846:Chromatography, Affinity; D004137:Dinitrochlorobenzene / Q000031:analogs & derivatives* / Q000378:metabolism; D005982:Glutathione Transferase / Q000302:isolation & purification / Q000378:metabolism*; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D051381:Rats; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
chemical_list: D004137:Dinitrochlorobenzene; D007527:Isoenzymes; D005982:Glutathione Transferase