Valine 10 may act as a driver for product release from the active site of human glutathione transferase P1-1

Abstract

We have probed the electrophilic binding site (H-site) of human glutathione transferase P1-1 through mutagenesis of two valines, Val 10 and Val 35, into glycine and alanine, respectively. These two residues were previously shown to be the only conformationally variable residues in the H-site and hence may play important roles in cosubstrate recognition and/or product dissociation. Both of these mutant enzymes have been expressed in Escherichia coli and purified and their kinetic properties characterized. The results demonstrate that Va135 Ala behaves similarly to wild, type, whereas Val10Gly exhibits a strong decrease of kcat and kcat/Kmcosub toward two selected cosubstrates: ethacrynic acid..