CHAC2 is a cytosolic γ-glutamylcyclotransferase that catalyzes the specific cleavage of glutathione into 5-oxo-L-proline and Cys-Gly dipeptide 12. The enzyme shows high specificity for reduced glutathione and does not act on other γ-glutamyl peptides or oxidized glutathione 2. CHAC2 exhibits 10-20-fold lower catalytic efficiency compared to its paralog CHAC1, with a Km of 3.7 mM and kcat of 15.9 min⁻¹ for human CHAC2 2. The protein contains a flexible loop that functions as a gate for glutathione specificity, with Glu74 and Glu83 being crucial for enzyme conformation and activity 1. CHAC2 plays complex roles in cancer biology, acting as a tumor suppressor in gastric and colorectal cancers by inducing apoptosis and autophagy through unfolded protein response 3, but promoting breast cancer cell proliferation 14. The enzyme is essential for glutathione homeostasis in human embryonic stem cells, where it competes with CHAC1 to prevent glutathione degradation and maintain self-renewal 5. CHAC2 is constitutively expressed and involved in continuous basal turnover of cytosolic glutathione 26.