title: Glutathione S-transferase Pi has at least three distinguishable xenobiotic substrate sites close to its glutathione-binding site.
abstract: Benzyl isothiocyanate (BITC), present in cruciferous vegetables, is an efficient substrate of human glutathione S-transferase P1-1 (hGST P1-1). BITC also acts as an affinity label of hGST P1-1 in the absence of glutathione, yielding an enzyme inactive toward BITC as substrate. As monitored by using BITC as substrate, the dependence of k of inactivation (K(I)) of hGST P1-1 on [BITC] is hyperbolic, with K(I) = 66 +/- 7 microM. The enzyme incorporates 2 mol of BITC/mol of enzyme subunit upon complete inactivation. S-Methylglutathione and 8-anilino-1-naphthalene sulfonate (ANS) each yield partial protection against inactivation and decrease reagent incorporation, whereas S-(N-benzylthiocarbamoyl)glutathione or S-methylglutathione + ANS protects completely. Mapping of proteolytic digests of modified enzyme by using mass spectrometry reveals that Tyr(103) and Cys(47) are modified equally. S-Methylglutathione reduces modification of Cys(47), indicating this residue is at/near the glutathione binding region, whereas ANS decreases modification of Tyr(103), suggesting this residue is at/near the BITC substrate site, which is also near the binding site of ANS. The Y103F and Y103S mutant enzymes were generated, expressed, and purified. Both mutants handle substrate 1-chloro-2,4-dinitrobenzene normally; however, Y103S exhibits a 30-fold increase in K(m) for BITC and binds ANS poorly, whereas Y103F has a normal K(m) for BITC and K(d) for ANS. These results indicate that an aromatic residue at position 103 is essential for the binding of BITC and ANS. This study provides evidence for the existence of a novel xenobiotic substrate site in hGST P1-1, which can be occupied by benzyl isothiocyanate and is distinct from that of monobromobimane and 1-chloro-2,4 dinitrobenzene.
authors.affiliation: Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, USA.
authors.forename: Luis A
authors.initials: LA
authors.lastname: Ralat
authors.forename: Roberta F
authors.initials: RF
authors.lastname: Colman
mesh_terms: D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D005978:Glutathione / Q000378:metabolism*; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D008958:Models, Molecular; D009154:Mutation; D011489:Protein Denaturation; D013997:Time Factors; D015262:Xenobiotics / Q000378:metabolism*
chemical_list: D007527:Isoenzymes; D008024:Ligands; D015262:Xenobiotics; C496556:GSTP1 protein, human; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase; D005978:Glutathione