TY - JOUR
T1 - Mechanistic details of glutathione biosynthesis revealed by crystal structures of Saccharomyces cerevisiae glutamate cysteine ligase
AU - Biterova, Ekaterina I.
AU - Barycki, Joseph J.
PY - 2009/11/20
Y1 - 2009/11/20
N2 - 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 rate-limiting 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 ATP-independent 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 mutations.
AB - 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 rate-limiting 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 ATP-independent 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 mutations.
UR - http://www.scopus.com/inward/record.url?scp=70450285183&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=70450285183&partnerID=8YFLogxK
U2 - 10.1074/jbc.M109.025114
DO - 10.1074/jbc.M109.025114
M3 - Article
C2 - 19726687
AN - SCOPUS:70450285183
SN - 0021-9258
VL - 284
SP - 32700
EP - 32708
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 47
ER -