Biochemistry, Department of


Date of this Version



The Journal of Biological Chemistry VOL. 284, NO. 47, pp. 32700–32708, November 20, 2009


© 2009 by The American Society for Biochemistry and Molecular Biology, Inc.


Glutathione is a thiol-disulfide exchange peptide critical for

buffering oxidative or chemical stress, and an essential cofactor

in several biosynthesis and detoxification pathways. The ratelimiting

step in its de novo biosynthesis is catalyzed by glutamate

cysteine ligase, a broadly expressed enzyme for which limited

structural information is available in higher eukaryotic species.

Structural data are critical to the understanding of clinical glutathione

deficiency, as well as rational design of enzyme modulators

that could impact human disease progression. Here, we

have determined the structures of Saccharomyces cerevisiae glutamate

cysteine ligase (ScGCL) in the presence of glutamate and

MgCl2 (2.1 Å; R = 18.2%, Rfree = 21.9%), and in complex with

glutamate, MgCl2, and ADP (2.7 Å ; R = 19.0%, Rfree = 24.2%).

Inspection of these structures reveals an unusual binding pocket

for the α-carboxylate of the glutamate substrate and an ATPindependent

Mg2+ coordination site, clarifying the Mg2+

dependence of the enzymatic reaction. The ScGCL structures

were further used to generate a credible homology model of the

catalytic subunit of human glutamate cysteine ligase (hGCLC).

Examination of the hGCLC model suggests that post-translational

modifications of cysteine residues may be involved in the

regulation of enzymatic activity, and elucidates the molecular

basis of glutathione deficiency associated with patient hGCLC