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

Abstract

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 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 with a dissociation constant (K d ) of 59 ؎ 17 M while the aflatoxinglutathione conjugate bound the enzyme with a K d of 0.86 ؎ 0.19 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 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.Glutathione S-transferases (GSTs; EC 2.5. 1.18) 1 are a family of broad specificity detoxification enzymes which catalyze the conjugation of glutathione to xenobiotics and endogenous substrates with electrophilic functional groups. Conjugation of reactive substrates with glutathione by GST serves primarily to prevent deleterious reactions with nucleophilic centers in cellular macromolecules. As first shown by Benson et al. (1), overexpression of GST often provides protection against the effects of electrophile exposure. In contrast, depletion of glutathione can increase cellular damage caused by GST substrates (2). In vertebrates, the cytosolic GSTs consist of four classes of isoenzymes: alpha, mu, pi, and theta, which all have similar three-dimensional structures (3-6). The active form of GST is a dimer, either a homodimer or a heterodimer, composed of two subunits from the same class (7,8).Structural and functional studies have clearly shown that the GST homodimer has two identical binding sites (one per subunit) which act independently rather than cooperatively. Structures have been solved for numerous crystals of GST isoenzymes complexed with different GST products or product analogs in the active site. These crystals do not display any significant structural differences between the two subunits in the dimer, which might suggest differences in activity of the subunits (4, 5, 9, 10). In addition, many of these structures clearly show two products bound per dimer, one in the active site of each subunit (4, 5, 9 -11). Ligand binding experiments have shown noncooperative substrate and product binding with a stoichiometry of two ligands per dimer (12). In additio...

Development of a microscopic platform for real-time monitoring of biomolecular interactions

“…Figure 3B shows the time course of the binding of the Cy3-GSH to GST at several concentrations (50-500 nM) of Cy3-GSH. The K D value of Cy3-GSH to GST obtained from these results was 1.3 ‫ן‬ 10 ‫6מ‬ (M), which roughly agrees with those obtained in previous studies using 35 S-labeled GSH or other GSH derivatives (Jakobson et al 1979;McHugh et al 1996). Thus, the microbead-based array platform enabled us to successfully monitor the binding of lowmolecular-weight molecules.…”

Aflatoxin B1 and sulphobromophthalein binding to the dimeric human glutathione S‐transferase A1‐1 : a fluorescence spectroscopic analysis

“…2), indicates that 1.1 mol of aflatoxin B1 binds per mol of protein. It has previously been shown that one molecule of an aflatoxinϪglutathione conjugate binds per protein dimer in the mouse GST A3-3 [19]. Furthermore, at aflatoxin B1 concentrations less than 100 µM, there is no inhibition of the protein's enzymatic activity with respect to CDNB as the electrophilic substrate [aflatoxin B1 concentrations exceeding 100 µM interfere with the activity assay due to its high absorption at 340 nm (Fig.…”

“…The human, rat and mouse isoenzymes are closely related in amino acid sequence (sequence similarity Ͼ 75%) and these differences in inhibition (or lack thereof) may reflect subtle variances in the hydrophobic-electrophilic and/or non-substrate ligand binding sites. While the human and rat isoenzymes exhibit very low catalytic efficiencies with respect to the conjugation reaction between glutathione and the aflatoxin B1 exo-epoxide (90 M Ϫ1 s Ϫ1 for the human enzyme; 300 M Ϫ1 s Ϫ1 for the rat enzyme [37]), it has been reported that the mouse enzyme shows a significantly higher activity toward the aflatoxin B1 exo-8,9-epoxide than either of the other two enzymes [19].…”

“…Alternatively, non-substrate ligand binding may cause no inhibition of the protein's enzymatic activity whatsoever (for example, the binding of praziquantel to S. japonicum GST does not result in enzymatic inhibition of the protein [36]). Even though the class alpha GSTs are capable of conjugating glutathione to aflatoxin exo-8,9-epoxide [19], aflatoxin B1 does not appear to inhibit human GST A1-1 enzymatic activity with respect to CDNB as the electrophilic substrate. Although an aflatoxinϪglutathione conjugate was found to inhibit activity of the mouse GST A3-3 toward CDNB, it did not inhibit the activity of rat GST A3-3, even though both of these isoenzymes have similar CDNB-specific activities [19].…”

“…Even though the class alpha GSTs are capable of conjugating glutathione to aflatoxin exo-8,9-epoxide [19], aflatoxin B1 does not appear to inhibit human GST A1-1 enzymatic activity with respect to CDNB as the electrophilic substrate. Although an aflatoxinϪglutathione conjugate was found to inhibit activity of the mouse GST A3-3 toward CDNB, it did not inhibit the activity of rat GST A3-3, even though both of these isoenzymes have similar CDNB-specific activities [19]. The human, rat and mouse isoenzymes are closely related in amino acid sequence (sequence similarity Ͼ 75%) and these differences in inhibition (or lack thereof) may reflect subtle variances in the hydrophobic-electrophilic and/or non-substrate ligand binding sites.…”