Catalyzes the formation of an amide bond between glutathione (GSH) and spermidine coupled with hydrolysis of ATP; also catalyzes the opposing reaction, i.e. the hydrolysis of glutathionylspermidine (Gsp) back to glutathione and spermidine. The amidase active site can also hydrolyze Gsp-disulfide (Gsp-S-S-Gsp) to Gsp-SG and Gsp S-thiolated proteins (GspSSPs) to GSH S-thiolated protein (GSSPs). Likely acts synergistically with glutaredoxin to regulate the redox environment of E.coli and defend against oxidative damage. In vitro, the amidase active site also catalyzes hydrolysis of amide and ester derivatives of glutathione (e.g. glutathione ethyl ester and glutathione amide) but lacks activity toward acetylspermidine (N1 and N8) and acetylspermine (N1). ATP + glutathione + spermidine = ADP + glutathionylspermidine + H(+) + phosphate glutathionylspermidine + H2O = glutathione + spermidine When exposed to oxidative stress, Gsp amidase activity is transiently inhibited in vivo by oxidation of the catalytic Cys-59 thiol to sulfenic acid; this modification does not affect Gsp synthetase activity. Gsp amidase activity is negatively autoregulated by the Gsp synthetase domain, and is activated by the Gsp synthetase substrates, GSH and ATP-Mg(2+); the occupancy of the synthetase active site may initiate communication through the protein as manifest by the release of inhibition of the amidase activity. A tetrahedral phosphonate analog of glutathionylspermidine, designed as a mimic of the proposed tetrahedral intermediate for either reaction, inhibits the synthetase activity (Ki of 10 uM) but does not inhibit the amidase activity. Amidase activity is inhibited by iodoacetamide in vitro. kcat is 7 sec(-1) for Gsp synthetase activity at pH 6.8 and 2.1 sec(-1) for Gsp amidase activity at pH 7.5. Optimum pH is around 6.8 for Gsp synthetase activity. Sulfur metabolism; glutathione metabolism. Amine and polyamine metabolism; spermidine metabolism. Homodimer. Expression level is unaffected by H(2)O(2); however Gsp rapidly accumulates in E.coli in the presence of H(2)O(2). The two activities reside in distinct domains (N-terminal amidase and C-terminal synthetase). The two domains expressed independently are folded and functional; liberation of the amidase domain from the synthetase domain highly activates the amidase activity. Oxidation of Cys-59 to sulfenic acid during oxidative stress selectively inhibits the amidase activity which leads to a rapid increase in the amounts of intracellular Gsp and Gsp S-thiolated proteins (GspSSPs). Cells lacking this gene do not produce Gsp under anaerobic conditions. Cells lacking both this gene and glutaredoxin (grxA or grxB) become hypersensitive to H(2)O(2); they are even more susceptible to oxidative damage than the single mutant lacking glutaredoxin only. Gsp forms mixed disulfides with the thiols of a variety of E.coli proteins. These mixed disulfides represent a previously uncharacterized type of post-translational modification. The level of these proteins is increased by oxidative stress, which implies that Gsp might protect protein thiols against irreversible oxidation (PubMed:20530482). No metal ion is required for the amidase activity. Gsp hydrolysis to GSH and spermidine proceeds with formation of a glutathionyl acyl-enzyme intermediate, utilizing a cysteine residue as the catalytic nucleophile (PubMed:9398217). For Gsp synthesis, GSH is likely phosphorylated at one of two GSH-binding sites to form an acylphosphate intermediate that then translocates to the other site for subsequent nucleophilic addition of spermidine (PubMed:17124497). In the C-terminal section; belongs to the glutathionylspermidine synthase preATP-grasp family.