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Volume 271, Number 44, Issue of November 1, 1996 pp. 27470-27474
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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

(Received for publication, August 6, 1996, and in revised form, August 27, 1996)

Thomas E. McHugh ^§ , William M. Atkins ^¶ , Jagdish K. Racha ^¶ , Kent L. Kunze ^¶ and David L. Eaton ^§

From the  Center for Ecogenetics and Environmental Health, ^§ Department of Environmental Health, School of Public Health and Community Medicine, and ^¶ Department of Medicinal Chemistry, School of Pharmacy, University of Washington, Seattle, Washington 98195

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 (K_d) of 59 ± 17 µM while the aflatoxin-glutathione conjugate bound the enzyme with a K_d of 0.86 ± 0.19 µM. Glutathione competitively inhibited binding of AFB-GSH to mGSTA3-3 with a K_i 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.

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