Formation of the 1-(S-glutathionyl)-2,4,6-trinitrocyclohexadienate anion at the active site of glutathione S-transferase: evidence for enzymic stabilization of .sigma.-complex intermediates in nucleophilic aromatic substitution reactions

Abstract

Formation of the Meisenheimer complex or .sigma.-complex [1-(S-glutathionyl)-2,4,6-trinitrocyclohexadienate] between glutathione (GSH) and 1,3,5-trinitrobenzene (TNB) can be observed at the active sites of isoenzymes 3-3 and 4-4 of rat liver GSH transferase. The spectroscopic properties (UV-visible and CD) of the enzyme-bound .sigma.-complex are consistent with a 1:1 complex in an asymmetric environment. Competitive inhibitors which occupy the GSH binding site (e.g., .gamma.-L-glutamyl-D,L-2-aminomalonylglycine) inhibit complex formation. The apparent formation constants of the .sigma.-complex (M) with enzyme-bound GSH (E .cntdot. EGS- + TNB .dblarw. E .cntdot. M) at pH 7.5 are 5 .times. 104 M-1 and 7 .times. 102 M-1 for isoenzymes 3-3 and 4-4, respectively. Both values are much greater than that in aqueous solution (GS- + TNB .dblarw. M), where Kf aromatic substitutions, a fact that appears to correlate with the ability of each enzyme to stabilize the .sigma.-complex. The pH dependence of Kf(app) for isoenzyme 3-3 is used to probe the ionization behavior of enzyme-bound GSH. The results are consistent with a double-ionization scheme (e.g., H+E .cntdot. GSH .dblarw. H+E .cntdot. GS- .dblarw.E .cntdot. GS-) with pK''s of 5.7 and 7.6, which are assigned to the thiol pK and the pK of a protonated base in the active site, respectively. Formation of the .sigma.-complex is also observed in single crystals of isoenzyme 3-3, providing a clear demonstration of the chemical competence of the crystallized enzyme. The results are discussed with respect to catalytic efficiency and the ability of the enzyme to stabilize .sigma.-complex intermediates in nucleophilic aromatic substitution reactions.