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 glutathi one and the aflatoxin-glutathione conjugate) to mouse glutathione S-tr ansferase A3-3 (mGSTA3-3) has been measured using equilibrium dialysis and a direct fluorescence quenching technique, As expected, p-nitrobe nzyl glutathione was found to bind with a stoichiometry of 2.24 +/- 0. 17 mol/mol of dimeric enzyme. However, the much larger aflatoxin-gluta thione conjugate, ,9-dihydro-S-(S-glutathionyl)-9-hydroxyl-aflatoxin B -1 (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 wi th a dissociation constant (K-d) of 59 +/- 17 mu M while the aflatoxin -glutathione conjugate bound the enzyme with a K-d of 0.86 +/- 0.19 mu M. 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 enzy me active site. Although AFB-GSH bound to mGSTA3-3 with a stoichiometr y of 1 mol/mol of dimeric enzyme, AFB-GSH completely inhibited activit y toward 1-chloro-2,4-dinitrobenzene, indicating that AFB-GSH binding to one active site alters affinity for 1-chloro-2,4-dinitrobenzene ill the active site of the other subunit. To our knowledge, this is the f irst report of a glutathione S-transferase reaction product which bind s to the enzyme with a stoichiometry of 1 mol/mol of dimer.