STRUCTURES OF CLASS PI GLUTATHIONE-S-TRANSFERASE FROM HUMAN PLACENTA IN COMPLEX WITH SUBSTRATE, TRANSITION-STATE ANALOG AND INHIBITOR

Background: Glutathione S-transferases (GSTs) are detoxification enzym es, found in all aerobic organisms, which catalyse the conjugation of glutathione with a wide range of hydrophobic electrophilic substrates, thereby protecting the cell from serious damage caused by electrophil ic compounds. GSTs are classified into five distinct classes (alpha, m u, pi, sigma and theta) by their substrate specificity and primary str ucture. Human GSTs are of interest because tumour cells show increased levels of expression of single classes of GSTs, which leads to drug r esistance. Structural differences between classes of GST can therefore be utilised to develop new anti-cancer drugs. Many mutational and str uctural studies have been carried out on the mu and alpha classes of G ST to elucidate the reaction mechanism, whereas knowledge about the pi class is still limited. Results: We have solved the structures of the pi class GST hP1-1 in complex with its substrate, glutathione, a tran sition-state complex, the Meisenheimer complex, and an inhibitor, S-(p -bromobenzyl)-glutathione, and refined them to resolutions of 1.8 Angs trom, 2.0 Angstrom and 1.9 Angstrom respectively. All ligand molecules are well-defined in the electron density. In all three structures, an additionally bound N-morpholino-ethansulfonic acid molecule from the buffer solution was found. Conclusions: In the structure of the GST-gl utathione complex, two conserved water molecules are observed, one of which hydrogen bonds directly to the sulphur atom of glutathione and t he other forms hydrogen bonds with residues around the glutathione-bin ding site. These water molecules are absent from the structure of the Meisenheimer complex bound to GST, implicating that deprotonation of t he cysteine occurs during formation of the ternary complex which invol ves expulsion of the inner bound water molecule. The comparison of our structures with known mu class GST structures show differences in the location of the electrophile-binding site (H-site), explaining the di fferent substrate specificities of the two classes. Fluorescence measu rements are in agreement with the position of the N-morpholino-ethansu lfonic acid, close to Trp28, identifying a possible ligandin-substrate binding site.