2,4-DICHLOROPHENOXYACETIC ACID AND RELATED CHLORINATED COMPOUNDS INHIBIT 2 AUXIN-REGULATED TYPE-III TOBACCO GLUTATHIONE S-TRANSFERASES

Two auxin-inducible glutathione S-transferase (GST, EC 2.5.1.18) isozymes from tobacco (Nicotiana fabacum, White Burley) were partially characterized. GST1-1 and GST2-1 are members of a recently identified new type of plant GST isozymes that we will here refer to as type III. Both enzymes were active, with 1-chloro-2,4-dinitrobenzene as a substrate, when expressed in bacteria as fusion proteins. The apparent K-m for 1-chloro-2,4-dinitrobenzene was found to be 0.85 +/- 0.25 mM for GST1-1 and 0.20 +/- 0.15 mM for GST2-1. The apparent K-m for glutathione was similar for both enzymes, 0.40 +/- 0.15 mM. The in vitro activity of both enzymes could be inhibited by the synthetic auxin 2,4-dichlorophenoxyacetic acid, with an apparent K-i of 80 +/- 40 mu M for GST1-1 and 200 +/- 100 mu M for CST2-1. The CST1-1 was also inhibited by structurally related substances, such as 2,4-dichlorobenzoic acid, with a roughly similar K-i. The nonchlorinated structures benzoic acid and phenoxyacetic acid did not inhibit. p-Chloroisobutyric acid, or clofibric acid, an auxin-transport inhibitor, was found to be an active inhibitor as well. The strongest inhibitor identified, however, was a phenylacetic acid derivative, ethacrynic acid, which showed an apparent K-i of 5 +/- 5 mu M for both enzymes. This substance is a known inducer as well as a substrate of specific mammalian GSTs. The results presented here indicate that the type III plant GSTs might be involved in the metabolism or transport of chlorinated substances that are structurally related to auxins. The possibility that auxins are endogenous ligands or substrates for GSTs is discussed.