Mechanisms of glutathione conjugation

Conjugation reactions with glutathione have been reported for a vast number of compounds (see Table 1) and the catalytic mechanism has been clarified. For the GSTs, several mechanisms, including random, sequential and ping-pong have been proposed, but random binding order of substrates seems to prevail. However, from a physiological point of view glutathione binding should occur first because of the availability of glutathione in millimolar concentrations in the cells. This value is about three magnitudes larger than the dissociation constant between GSH and the enzymes (Wilce and Parker 1994).

Electrophilic centers necessary for GSH conjugation are found in arene-oxides, aliphatic and arylic halides, in a-b-unsaturated carbonyls, organonitro-esters and organic thiocyanates. Industrial substrates for GST are haloalkanes, chlorobenzenes, thiocarbamates, diphenyl­ethers, triazines, chloracetanilide. In animals the oxidants acrolein, several propenals, lipid hydro­peroxides, chlorambucil and fosfomycin are additional substrates.

For the xenobiotic binding there are in principle three different reaction types operating spontaneously or under catalysis by glutathione S-transferases (Figure 4, A-C).

Nucleophilic displacement of an alkyl or aryl halogen or a nitrogroup seems to be the most frequently observed step. Conjugation of the herbicides atrazine, pentachloronitrobenzene (PCNB), or methidathion are examples for this type of reaction. Halogens or nitrogroups of these molecules are soft electrophiles and react readily with the GSH. In fact, the standard enzymological assays for glutathione S-transferase activity use 1-chloro-2,4-dinitrobenzene (CDNB) or 1,2-dinitro-4-chloro­benzene (DCNB) as substrates (Figure 5).

Figure 4. Mechanisms for the conjugation of xenobiotics with glutathione (from Lamoureux & Rusness 1993 modified).

Also to the substitution reactions belongs the detoxification of diphenylether herbicides (e.g. fluorodifen, fenoxaprop-ethyl). Here, an ether bond is cleaved and substituted by the thiolate. The phenyl residue is released and may be further conjugated with sugars.

Addition of the thiolate to carbon-carbon-double bonds has already been mentioned above. It is a special type of reactions on compounds with reactive carbon-carbon double bonds neighbored by an electron withdrawing group (Talalay et al. 1988). Conjugation of tridiphane or cinnamic acid may be examples for this type of reaction (Lamoureux & Rusness 1986, Diesperger & Sandermann 1979). The conjugation on these bonds is a so called Michael reaction and leads to a labile conjugate that may be sensitive to pH changes.

In plants, reactions with non carbon sites have scarcely been described, e.g. as been reported for diclofluanid (Schupahn et al. 1981), but they may still be significant.