Enzyme kinetics and substrate selectivities of rat glutathione S-transferase isoenzymes towards a series of new 2-substituted 1-chloro-4-nitrobenzenes.
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.
van der Aar
E M
EM
Department of Pharmacochemistry, Vrije Universiteit, Amsterdam, Netherlands.
Buikema
D
D
Commandeur
J N
JN
te Koppele
J M
JM
van Ommen
B
B
van Bladeren
P J
PJ
Vermeulen
N P
NP
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
D004137:Dinitrochlorobenzene; D007527:Isoenzymes; D005982:Glutathione Transferase