Binding of the aflatoxin-glutathione conjugate to mouse glutathione S-transferase A3-3 is saturated at only one ligand per dimer.
The binding of two different reaction products (p-nitrobenzyl glutathione and the aflatoxin-glutathione conjugate) to mouse glutathione S-transferase A3-3 (mGSTA3-3) has been measured using equilibrium dialysis and a direct fluorescence quenching technique. As expected, p-nitrobenzyl glutathione was found to bind with a stoichiometry of 2.24 +/- 0.17 mol/mol of dimeric enzyme. However, the much larger aflatoxin-glutathione conjugate, 8, 9-dihydro-8-(S-glutathionyl)-9-hydroxyl-aflatoxin B1 (AFB-GSH), was found to bind with a stoichiometry of 1.12 +/- 0.08 mol/mol of dimeric enzyme. p-Nitrobenzyl glutathione bound mGSTA3-3 with a dissociation constant (Kd) of 59 +/- 17 microM while the aflatoxin-glutathione conjugate bound the enzyme with a Kd of 0.86 +/- 0.19 microM. Glutathione competitively inhibited binding of AFB-GSH to mGSTA3-3 with a Ki of 1.5 mM, suggesting that AFB-GSH was binding to the enzyme active site. Although AFB-GSH bound to mGSTA3-3 with a stoichiometry of 1 mol/mol of dimeric enzyme, AFB-GSH completely inhibited activity toward 1-chloro-2, 4-dinitrobenzene, indicating that AFB-GSH binding to one active site alters affinity for 1-chloro-2,4-dinitrobenzene in the active site of the other subunit. To our knowledge, this is the first report of a glutathione S-transferase reaction product which binds to the enzyme with a stoichiometry of 1 mol/mol of dimer.
Center for Ecogenetics and Environmental Health, University of Washington, Seattle, Washington 98195, USA. deaton@u.washington.edu
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McHugh
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Eaton
D016604:Aflatoxin B1 / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D019281:Dimerization; D005978:Glutathione / Q000031:analogs & derivatives* / Q000378:metabolism*; D005982:Glutathione Transferase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013379:Substrate Specificity
D007527:Isoenzymes; D011994:Recombinant Proteins; C047285:8,9-dihydro-8-(S-glutathionyl)-9-hydroxyaflatoxin B1; D016604:Aflatoxin B1; D005982:Glutathione Transferase; D005978:Glutathione