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

Abstract

The binding interactions between dimeric human class alpha glutathione S-transferase A1-1 (GST A1-1) and aflatoxin B1 or sulphobromophthalein (BSP) were characterised. Aflatoxin B1 binds to GST A1-1 with a stoichiometry of 1.1 mol/mol of dimeric enzyme. The binding interaction, which can be described by a hyperbolic saturation isotherm (K d ϭ 8Ϯ 2 µM), does not induce major structural changes in the enzyme, nor does it inhibit enzymatic activity. The average distance between the single tryptophan residue (Trp20) of GST A1-1 and protein-bound aflatoxin B1 was calculated to be 22.7 Å by means of fluorescence resonance energy transfer. The aflatoxin-binding region, according to this calculated distance, was determined to be located in the dimer interface cleft near the crystallographic two-fold axis. Hill-plot analyses suggest that a positive co-operative interaction exists between BSP and the dimeric GST A1-1 (h ϭ 1.6 Ϯ0.1 ; K′ ϭ 14Ϯ0.6 µM). The binding of BSP induces a conformational change in the enzyme which is accompanied by a decrease in the molecular flexibility and in the solvent-accessible properties of the enzyme's Trp20 residue. Site-directed mutagenesis of Trp20 (Trp20→Phe) confirms that this residue is situated in the binding environment and although it is not essential for BSP binding, it is involved in the interaction. Furthermore, the structural change associated with BSP binding alters the hyperbolic character of the glutathione saturation curve. This indicates that there may also be a cooperative interaction between glutathione and BSP or that BSP binding induces asymmetric functioning of the two enzyme subunits so that they become unequal in catalytic activity.Keywords : glutathione S-transferase ; aflatoxin B1; sulphobromophthalein; co-operative binding.Glutathione S-transferases (GSTs) are a family of multifunctional enzymes found in all vertebrates, plants, insects, nematodes, yeast and aerobic bacteria. They constitute a complex supergene family that collectively metabolises a broad variety of potentially toxic xenobiotics and reactive endogenous compounds resulting from oxidative metabolism. GSTs are an integral part of the phase-I detoxification mechanism and primarily catalyse the nucleophilic addition of the thiol of reduced glutathione (γ-glutamyl-cysteinyl-glycine) to electrophilic centres in a wide variety of hydrophobic electrophiles, including alkyl and aryl halides, epoxides, quinones and activated alkenes [1]. The dimeric cytosolic GSTs exhibit multiple enzyme forms that, according to primary structure and molecular recognition, can be grouped into one of six species independent gene classes (alpha, kappa, mu, pi, theta and sigma). In addition to their catalytic activities, GSTs also function as ligand binding proteins and thereby facilitate the intracellular storage of a variety of hydrophobic non-substrate compounds, including hormones, metabolites and drugs [2]. The binding of these compounds to GSTs prevents the build up of apolar molecules at lipophilic sites such as membranes...

Thermodynamics of the ligandin function of human class Alpha glutathione transferase A1-1: energetics of organic anion ligand binding

“…The C-terminus of helix 9, also located near the dimer interface, is adjacent to the ANS site and modulates ANS binding [28]. Other non-substrate ligands that bind the V-shaped cleft include aflatoxin B " [12], oestradiol disulphate [13] and 5-o[2-[(acetyl)amino]ethyl]aminoq-naphthalene-1-sulphonic acid (AEDANS) [42]. The stoichiometry of binding apparently varies according to the size of the ligand.…”

“…glutathione conjugate in class Sigma GST with two type-1 subunits [10]. Fluorescence resonance energy transfer and affinity-labelling studies have also implicated the cleft in binding 8-anilino-1-naphthalene sulphonate (ANS) and aflatoxin B " [11,12], and steroid sulphates [13]. The dimeric structure of GSTs is therefore not only important for stabilizing the tertiary structures of GST subunits [14], it is also a requirement for the formation of non-substrate binding sites at the dimer interface.…”

“…This hydrophobic site is not related to the active site but may be involved in non-competitive inhibition of the enzyme via a ligand-induced conformational change in helix 8, the C-terminus of which is close to the active site. This ligandin site might be involved in binding bromosulphophthalein [12,17,19,20], bilirubin [21], fatty acids [22] and 2-hydroxy-5-nitrobenzyl alcohol [23]. The stoichiometry of ligand binding to dimeric GSTs appears to depend on the size of the ligand and on whether it binds the intersubunit cleft (1 : 1 or 2 : 1) or the buffer binding site (2 : 1).…”

A Conserved N-capping Motif Contributes Significantly to the Stabilization and Dynamics of the C-terminal Region of Class Alpha Glutathione S-Transferases

“…ANS also competes with AFB 1 (61), whose exo-8,9-epoxide form binds the H-site of class Alpha GSTs where it is conjugated with GSH (62)(63)(64). A high affinitybinding site for ANS at or near the promiscuous H-site of hGST P1-1 has also been reported (65,66), in agreement with the competitive binding between ANS and BSP (67).…”

Characterization of the binding of 8-anilinonaphthalene sulfonate to rat class Mu GST M1-1

“…Crystallographic studies have shed light on the locations and properties of non-substrate binding sites on some GSTs; namely, the H-site of class Pi GST P1-1 [6], the dimer interface of the schistosomal Sj26GST [7] and class a Sigma GST [8], and other regions of GSTs to which sulfonate buffer compounds bind [6, 9, 10]. Other techniques employed to locate the position of and characterize non-substrate binding sites include fluorescence resonance energy transfer [11, 12], affinity labelling [13], inhibition kinetics [2, 14, 15], and isothermal titration calorimetry [16–19]. …”