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

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, in to glycine and alanine, respectively. These two residues were previously sh own to be the only conformationally variable residues in the H-site and hen ce may play important roles in cosubstrate recognition and/or product disso ciation. Both of these mutant enzymes have been expressed in Escherichia co li and purified and their kinetic properties characterized. The results dem onstrate that Va135Ala behaves similarly to wild-type, whereas Val10Gly exh ibits a strong decrease of k(cat) and k(cat)/K-m (cosub) toward two selecte d cosubstrates: ethacrynic acid and 1-chloro-2,4-dinitrobenzene. Pre-steady -state kinetic analysis of the GSH conjugation with ethacrynic acid shows t hat both wild-type and Val10Gly mutant enzymes exhibit the same rate-limiti ng step: the dissociation of product. However, in the Val10Gly mutant there is an increased energetic barrier which renders the dissociation of produc t more difficult. Similar results are found for the Val10Gly mutant with 1- chloro-2,4-dinitrobenzene as cosubstrate. With this latter cosubstrate, Val 10 also exerts a positive role in the conformational transitions of the te rnary complex before the chemical event. Crystallographic analysis of the V al10Gly mutant in complex with the inhibitor S-hexyl-GSH suggests that Val 10 optimally orientates products, thus promoting their exit from the active site.