Determination of a Binding Site for a Non‐Substrate Ligand in Mammalian Cytosolic Glutathione S‐Transferases by means of Fluorescence‐Resonance Energy Transfer

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Abstract
To determine the location of the non-substrate-ligand-binding region in mammalian glutathione S-transferases, fluorescence-resonance energy transfer was used to calculate distances between tryptophan residues and protein-bound 8-anilinonaphthalene 1-sulphonate (an anionic ligand) in the human class-α glutathione S-transferase, and in a human Trp28→Phe mutant class-π glutathione S-transferase. Distance values of 2.21 nm and 1.82 nm were calculated for the class-α and class-π enzymes, respectively. Since glutathione S-transferases bind one non-substrate ligandprotein dimer, the ligand-binding region, according to the calculated distances, is found to be located in the dimer interface near the twofold axis. This region is the same as that in which the parasitic helminth Schistosoma japonicum glutathione S-transferase binds praziquantel, a non-substrate drug used to treat schistosomiasis [ McTigue, M. A., Williams, D. R. & Tainer, J. A. (1995) J. Mol. Biol. 246, 21–271. Since the overall folding topology is conserved and certain features at the dimer interface are similar throughout the superfamily, it is reasonable to expect that all cytosolic glutathione S-transferases bind non-substrate ligands in the amphipathic groove at the dimer interface.

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