GlutathioneS-Transferase Pi Has at Least Three Distinguishable Xenobiotic Substrate Sites Close to Its Glutathione-binding Site

Luis A. Ralat, Roberta F. Colman
2004 Journal of Biological Chemistry  
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 M. The enzyme incorporates 2 mol of BITC/mol of enzyme subunit upon complete
more » ... ctivation. S-Methylglutathione and 8-anilino-1naphthalene 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.
doi:10.1074/jbc.m407445200 pmid:15347687 fatcat:magmj6fxkre2vgklj2q4ede35y