Functional Studies of Tyrosine 108 Residue in the Active Site of Human Glutathione 5-Transferase P1-1

摘要

To gain further insight on the relationship between structure and functions of glutathione-S-transferase(GST),the three tyrosine 108 mutants,Y108A,Y108F,and Y108W,of human GST Pl-1 were expressed in Escherichia coli and purified to electrophoretic homogeneity by affinity chromatography on immobilized GSH.The substitution of Tyr 108 with alanine resulted in significant decrease of the GSH-conjugation activity and the GSH peroxidase activity,but approximately 63% increase of steroid isomerase activity toward A5-androstene 3,17-dione.On the other hand,the substitution of Tyr 108 with phenylalanine resulted in decreases of kappa_(cat)and kappa_(cat)/K_m~(EPNP)by 2 orders of magnitude,suggesting that Tyr 108 residue of hGSTP 1-1 are considered to be important for the catalysis and the binding of the epoxide substrates.The substitution of Tyr 108 with tryptophan resulted in significant decreases of the specific activities toward EPNP,cumene hydroperoxide and A5-androstene 3,17-dione,but approximately 2-fold increase on the enzyme-catalyzed addition of GSH to DCNB.We conclude from these results that Tyr 108 in hGST Pl-1 plays very different roles depending upon the nature of the electrophilic substrates.