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, belo nging to three different classes, were examined for their GSH conjugat ing capacity towards 11 2-substituted 1-chloro-4-nitrobenzene derivati ves. Significant differences were found in their enzyme kinetic parame ters K-m, k(cat) and k(cat)/K-m.2. Substrates with bulky substituents on the ortho-position appeared to have high affinities (low K-m's) for the active site of the GST-isoenzymes, suggesting that there is suffi cient space in this area of the active site. A remarkably high K, (low affinity) was found for 2-chloro-5-nitropyridine towards all GST-isoe nzymes examined. 3. GST 3-3 catalysed the reaction between GSH and the substrates most efficiently (high k(cat)) compared with the other GST -isoenzymes. Moreover, GST 3-3 showed clear substrate selectivities to wards the substrates with a trifluoromethyl-, chlorine- and bromine-su bstituent. 1-Chloro-2,4-dinitrobenzene and 2-chloro-5-nitrobenzonitril e were most efficiently conjugated by ail 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-ni trobenzonitrile 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.