EVIDENCE FOR A GLUTATHIONYL-ENZYME INTERMEDIATE IN THE AMIDASE ACTIVITY OF THE BIFUNCTIONAL GLUTATHIONYLSPERMIDINE SYNTHETASE AMIDASE FROM ESCHERICHIA-COLI/

Glutathionylspermidine (Gsp) is a metabolite common to Escherichia col i and protozoal parasites of the Trypanosoma family. Though its role i n E. coli is unknown, Gsp is known to be an intermediate in the biosyn thesis of N',N-8-bis(glutathionyl)spermidine (trypanothione), a metabo lite unique to trypanosomatids that may allow the parasites to overcam e oxidative stresses induced by host defense mechanisms. The bifunctio nal Gsp-synthetase/amidase from E. coli catalyzes both amide bond form ation and breakdown between the N-1-amine of spermidine [N-(3-aminopro pyl)-1,4-diaminobutane] and the glycine carboxylate of glutathione (ga mma-Glu-Cys-Gly), with net hydrolysis of ATP [Bollinger et al, (1995) J. Biol, Chem. 270 (23), 14031-14041]. Synthetase and amidase activiti es reside in separate domains of the protein, and liberation of the am idase domain from the synthetase domain activates the amidase activity as much as 70-fold in k(cat)/K-m for a chromogenic substrate gamma-Gl u-Ala-Gly-pNA [Kwon et al., (1997) J. Biol. Chem. 272 (4), 2429-2436]. When substrates for the Gsp-synthetase activity are present (GSH, ATP -Mg2+), Gsp-amidase is highly activated (15-fold). We provide kinetic and mutagenesis evidence suggesting that the amidase operates by a nuc leophilic attack mechanism involving cysteine as the catalytic nucleop hile. Stopped-flow studies on the 25 kDa Gsp-amidase fragment and the 70 kDa full-length Gsp-synthetase/amidase with gamma-Glu-Ala-Gly-ONp d emonstrate burst kinetics characteristic of a covalent acyl-enzyme int ermediate. Studies using various group-specific protease inhibitors, s uch as iodoacetamide, suggest an active-site cysteine or histidine as being relevant to amidase activity, and site-directed mutagenesis indi cates that Cys-59 is essential for amidase activity.