In search of potential residues involved in co-substrate recognition or product dissociation we have probed the electrophilic binding site (H-site) of human placental glutathione transferase (GST P1-1) by mutating two valines (Val 10 and Val 35) into glycine and alanine, respectively. The results demonstrate that Val35Ala behaves similar to wild type, whereas Val10Gly exhibits a strong decrease of kcat towards two selected co-substrates, ethacrynic acid and 1-chloro-2,4-dinitrobenzene. Kinetic, spectroscopic and crystallographic analysis of the Val10Gly mutant enzyme indicate that Val 10, located on the floor of the H-site, may orient products and help them to leave the active site.
|Number of pages||4|
|Publication status||Published - 28 Feb 2001|
- Catalytic mechanism
- Glutathione transferase
- Site-directed mutagenesis