Binding of the Aflatoxin-Glutathione Conjugate to Mouse Glutathione S-Transferase A3-3 Is Saturated at Only One Ligand per Dimer

• Published in: The Journal of biological chemistry 1996-11, Vol.271 (44), p.27470-27474
• Main Authors: McHugh, Thomas E., Atkins, William M., Racha, Jagdish K., Kunze, Kent L., Eaton, David L.
• Format: Article
• Language: English
• Subjects: Aflatoxin B1 - analogs & derivatives; Aflatoxin B1 - metabolism; Animals; Binding Sites; Cloning, Molecular; Dimerization; Glutathione - analogs & derivatives; Glutathione - metabolism; Glutathione Transferase - chemistry; Glutathione Transferase - isolation & purification; Glutathione Transferase - metabolism; Isoenzymes - chemistry; Isoenzymes - isolation & purification; Isoenzymes - metabolism; Kinetics; Mice; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Spectrometry, Fluorescence; Spectrophotometry; Substrate Specificity
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.271.44.27470

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 µM while the aflatoxin-glutathione conjugate bound the enzyme with a Kd of 0.86 ± 0.19 µM. 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.