Structural Basis for Feedback and Pharmacological Inhibition of Saccharomyces cerevisiae Glutamate Cysteine Ligase

Abstract

Structural characterization of glutamate cysteine ligase (GCL), the enzyme that catalyzes the initial, rate-limiting step in glutathione biosynthesis, has revealed many of the molecular details of substrate recognition. To further delineate the mechanistic details of this critical enzyme, we have determined the structures of two inhibited forms of Saccharomyces cerevisiae GCL (ScGCL), which shares significant sequence identity with the human enzyme. In vivo, GCL activity is feedback regulated by glutathione. Examination of the structure of ScGCL-glutathione complex (2.5 Å ; R ‫؍‬ 19.9%, R free ‫؍‬ 25.1%) indicates that the inhibitor occupies both the glutamate-and the presumed cysteine-binding site and disrupts the previously observed Mg 2؉ coordination in the ATP-binding site. L-Buthionine-S-sulfoximine (BSO) is a mechanism-based inhibitor of GCL and has been used extensively to deplete glutathione in cell culture and in vivo model systems. Inspection of the ScGCL-BSO structure (2.2 Å ; R ‫؍‬ 18.1%, R free ‫؍‬ 23.9%) confirms that BSO is phosphorylated on the sulfoximine nitrogen to generate the inhibitory species and reveals contacts that likely contribute to transition state stabilization. Overall, these structures advance our understanding of the molecular regulation of this critical enzyme and provide additional details of the catalytic mechanism of the enzyme. Glutamate cysteine ligase (GCL)2 catalyzes the initial and rate-limiting step of glutathione biosynthesis (1, 2). The ATPdependent mechanism proceeds via a ␥-glutamylphosphate intermediate (2-4), with a subsequent nucleophilic attack by the ␣-amino group of L-cysteine to produce ␥-glutamylcysteine (1, 2). There are three distinct families of GCL enzymes: ␥-proteobacteria (Group 1), nonplant eukaryotes (Group 2), and ␣-proteobacteria and plants (Group 3) (5). Despite low sequence conservation between these groups (typically Ͻ10% sequence identity), all of the GCL appear to use this general catalytic mechanism. The resulting ␥-glutamylcysteine is coupled to L-glycine by glutathione synthetase (1) in an analogous reaction to generate reduced GSH, an abundant cellular reducing agent.GCL activity is tightly modulated by free L-cysteine availability (6), transcriptional regulation (7), and post-translational modifications (8). In addition, GCL is feedback regulated by the end product, glutathione (9). Glutathione inhibits GCL competitively with respect to L-glutamate, suggesting that the two binding sites are coincident (9). In heterodimeric GCL, such as the Drosophila, rat, and human enzymes, binding of the modifier subunit relieves feedback inhibition both by increasing the K i for glutathione and decreasing the K m for glutamate (10 -13). Further studies with glutathione analogues such as ophthalmic acid, S-methylglutathione, and GSSG have demonstrated that the free thiol group of glutathione is necessary for maximal inhibition (1, 9). However, the precise mode of glutathione binding has not been described.The central role of GCL in glutathione homeosta...

Glutathione Degradation Is a Key Determinant of Glutathione Homeostasis

“…Therefore, inhibition of ␥-GCS may increase Cys concentration and restore the normal rate of GSH synthesis. Another explanation could simply be the fact that GSH is a competitive inhibitor of ␥-GCS with respect to Glu (32). Considering that the K m for Glu is 1.2 mM (32) and that in yeast Glu intracellular concentration is more than 20 mM (45,46), doubling GSH concentration from 3 to 6 mM may not significantly decrease ␥-GCS activity.…”

“…An important result of this work is that in vivo, a 2-fold increase of GSH does not seem to affect GSH synthesis, although based on in vitro experiments, GSH is considered as an inhibitor of ␥-GCS in many organisms (28 -30), including S. cerevisiae (32). In yeast, the K i value for GSH, 2.1 mM (32), is close to the intracellular concentration of GSH (Refs.…”

“…In yeast, the K i value for GSH, 2.1 mM (32), is close to the intracellular concentration of GSH (Refs. 15 and 22) and this work).…”

“…Transposing the metabolic model to a mutant strain without changing any flux in the GSH cycle (except the one put to 0) postulates that changes in GSH concentration do not affect the GSH synthesis rate. This is not trivial because in vitro experiments have shown that GSH is an inhibitor of ␥-GCS in organisms as diverse as Escherichia coli (28), rats (28,29), Arabidopsis thaliana (30), and yeasts (31,32), and it is generally admitted that S. cerevisiae ␥-GCS is also subjected to such a feedback inhibition in vivo (33).…”

Expression of Bacterial GshF in Pichia pastoris for Glutathione Production

“…GshF also has other potential advantages over the conventional ␥-GCS/GS systems when used for GSH production. First, the feedback inhibition of ␥-GCS activity by GSH is considered a physiological mechanism to prevent the overaccumulation of GSH (1,2). GshF from S. agalactiae and Streptococcus thermophilus was shown to be insensitive to an increased concentration of GSH (9,11).…”