Aflatoxin B1 and sulphobromophthalein binding to the dimeric human glutathione S-transferase A1-1: a fluorescence spectroscopic analysis.
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 (Kd = 8+/-2 microM), 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 A 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 = 1.6+/-0.1; K' = 14+/-0.6 microM). 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.
Sluis-Cremer
N
N
Department of Biochemistry, University of the Witwatersrand, Johannesburg, South Africa.
Wallace
L
L
Burke
J
J
Stevens
J
J
Dirr
H
H
D016604:Aflatoxin B1 / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology; D019281:Dimerization; D004735:Energy Transfer; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013448:Sulfobromophthalein / Q000378:metabolism*; D014364:Tryptophan / Q000378:metabolism
D007527:Isoenzymes; D013448:Sulfobromophthalein; D014364:Tryptophan; D016604:Aflatoxin B1; D005982:Glutathione Transferase; C119995:glutathione S-transferase alpha