In enzymology, a glutathionylspermidine amidase (EC 3.5.1.78) is an enzyme that catalyzes the chemical reaction

glutathionylspermidine + H₂O ${\displaystyle \rightleftharpoons }$ glutathione + spermidine

Thus, the two substrates of this enzyme are glutathionylspermidine and H₂O, whereas its two products are glutathione and spermidine.

This enzyme belongs to the family of hydrolases, those acting on carbon-nitrogen bonds other than peptide bonds, specifically in linear amides. The systematic name of this enzyme class is gamma-L-glutamyl-L-cysteinyl-glycine:spermidine amidase. This enzyme is also called glutathionylspermidine amidohydrolase (spermidine-forming). This enzyme participates in glutathione metabolism.

As of late 2007, 5 structures have been solved for this class of enzymes, with PDB accession codes 2IO7, 2IO8, 2IO9, 2IOA, and 2IOB.

• Bollinger JM, Kwon DS, Huisman GW, Kolter R, Walsh CT (1995). "Glutathionylspermidine metabolism in Escherichia coli. Purification, cloning, overproduction, and characterization of a bifunctional glutathionylspermidine synthetase/amidase". J. Biol. Chem. 270 (23): 14031–41. doi:10.1074/jbc.270.23.14031. PMID 7775463.