Structural analysis on the ligand specificity of γ-glutamylcysteine synthetase

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γ-Glutamylcysteine synthetase (γGCS), a rate-limiting enzyme in glutathione biosynthesis, plays a central role in glutathione homeostasis and is a target for development of potential therapeutic agents against parasites and cancer.We have determined the crystal structures of E. coli γGCS complexed with a sulfoximine-based transition-state analog inhibitor and a classical inhibitor, buthionine sulfoximine (BSO), at resolutions of 2.1 and 2.3 A, respectively. The cysteine-binding site was identified; it is formed inductively at the transition state. In the unliganded structure, an openspace exists around the representative cysteine-binding site and is probably responsible for the competitive binding of glutathione. Upon binding of the transition-state analog, the side chains of Tyr-241 and Tyr-300 turn, forming a hydrogen-bonding triad with the carboxyl group of the inhibitors cysteine moiety, allowing this moiety to fit tightly into the cysteine-binding site with concomitant accommodation of its side chain into a shallow pocket. This movement is caused by a conformational change of a switch loop (residues 240-249). The side chain of Tyr-300 gives no electron density peak in the complex with BSO that has no carboxyl group essential to the formation of the hydrogen-bonding triad.Based on this crystal structure, the cysteine-binding sites of mammalian and parasitic γGCSs were predicted by multiple sequence alignment, although no significant sequence identity exists between the E. coli γGCS and its eukaryotic homologues. The identification of this cysteine-binding site provides important information for the rational design of novel γGCS inhibitors.