Abstract
Glutathione degradation plays an important role in glutathione and redox homeostasis, and thus it is imperative to understand the enzymes and the mechanisms involved in glutathione degradation in detail. We describe here ChaC2, a member of the ChaC family of γ-glutamylcyclotransferases, as an enzyme that degrades glutathione in the cytosol of mammalian cells. ChaC2 is distinct from the previously described ChaC1, to which ChaC2 shows ∼50% sequence identity. Human and mouse ChaC2 proteins purified in vitro show 10-20-fold lower catalytic efficiency than ChaC1, although they showed comparable Km values (Km of 3.7 ± 0.4 mm and kcat of 15.9 ± 1.0 min-1 toward glutathione for human ChaC2; Km of 2.2 ± 0.4 mm and kcat of 225.2 ± 15 min-1 toward glutathione for human ChaC1). The ChaC1 and ChaC2 proteins also shared the same specificity for reduced glutathione, with no activity against either γ-glutamyl amino acids or oxidized glutathione. The ChaC2 proteins were found to be expressed constitutively in cells, unlike the tightly regulated ChaC1. Moreover, lower eukaryotes have a single member of the ChaC family that appears to be orthologous to ChaC2. In addition, we determined the crystal structure of yeast ChaC2 homologue, GCG1, at 1.34 Å resolution, which represents the first structure of the ChaC family of proteins. The catalytic site is defined by a fortuitous benzoic acid molecule bound to the crystal structure. The mechanism for binding and catalytic activity of this new enzyme of glutathione degradation, which is involved in continuous but basal turnover of cytosolic glutathione, is proposed.
Highlights
Glutathione degradation plays an important role in glutathione and redox homeostasis, and it is imperative to understand the enzymes and the mechanisms involved in glutathione degradation in detail
When mouse ChaC1 was compared with human ChaC1, there was an 88% sequence identity, and mouse ChaC2 compared with human ChaC2 reveals an ϳ94% identity between the proteins
The phylogenetic tree strongly suggests that the ChaC family consists of two different branches represented by the ChaC1 and ChaC2 proteins (Fig. 1)
Summary
Glutathione degradation plays an important role in glutathione and redox homeostasis, and it is imperative to understand the enzymes and the mechanisms involved in glutathione degradation in detail. We describe here ChaC2, a member of the ChaC family of ␥-glutamylcyclotransferases, as an enzyme that degrades glutathione in the cytosol of mammalian cells. The second pathway involves the ChaC1 enzyme, a member of the ChaC family of ␥-glutamylcyclotransferases that can degrade glutathione to 5-oxoproline and Cys-Gly [10] and is induced under conditions of ER stress in the cell [10, 18]. The mouse and human ChaC1 function as glutathione-degrading enzymes [10, 19], the function of the mammalian ChaC2 proteins has not been explored. We have solved the crystal structure of the ChaC2 homologue in yeast This is the first crystal structure to be determined for any member of the ChaC family of proteins and provides insights into the mechanism of action of ChaC proteins
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