Abstract
Glutathionylspermidine (GSP) synthetases of Trypanosomatidae and Escherichia coli couple hydrolysis of ATP (to ADP and Pi) with formation of an amide bond between spermidine (N-(3-aminopropyl)-1,4-diaminobutane) and the glycine carboxylate of glutathione (gamma-Glu-Cys-Gly). In the pathogenic trypanosomatids, this reaction is the penultimate step in the biosynthesis of the antioxidant metabolite, trypanothione (N1,N8-bis-(glutathionyl)spermidine), and is a target for drug design. In this study, GSP synthetase was purified to near homogeneity from E. coli B, the gene encoding it was isolated and sequenced, the enzyme was overexpressed and purified in quantity, and the recombinant enzyme was characterized. The 70-kDa protein was found to have an unexpected second catalytic activity, glutathionylspermidine amide bond hydrolysis. Thus, the bifunctional GSP synthetase/amidase catalyzes opposing amide bond-forming and -cleaving reactions, with net hydrolysis of ATP. The synthetase activity is selectively abrogated by proteolytic cleavage 81 residues from the C terminus, suggesting that the two activities reside in distinct domains (N-terminal amidase and C-terminal synthetase). Proteolysis at this site is facile in the absence of substrates, but is inhibited in the presence of ATP, glutathione, and Mg2+. A series of analogs was used to probe the spermidine-binding site of the synthetase activity. The activity of diaminopropane as a substrate, inactivity of the C4-C8 diaminoalkanes, and greater loss of specificity for analogs modified in the 3-aminopropyl moiety than for those modified in the 4-aminobutyl moiety indicate that the enzyme recognizes predominantly the diaminopropane portion of spermidine and corroborate N-1 (the aminopropyl N) as the site of glutathione linkage (Tabor, H. and Tabor, C. W. (1975) J. Biol. Chem. 250, 2648-2654). Trends in Km and kcat for a set of difluorosubstituted spermidine derivatives suggest that the enzyme may bind the minor, deprotonated form of the amine nucleophile.
Highlights
Glutathionylspermidine (GSP) synthetases of Trypanosomatidae and Escherichia coli couple hydrolysis of ATP with formation of an amide bond between spermidine (N-(3-aminopropyl)-1,4-diaminobutane) and the glycine carboxylate of glutathione (y-Glu-Cys-Gly)
All enzyme activity was found in the wash fractions, and these were pooled and loaded onto a 2.6 X 12-cm hydroxylapatite column equilibrated in 20 mM potassium phosphate buffer, pH 6.8, The column was developed with a 20-ml gradient of 20-68 mM potassium Pi' with a 300-ml gradient of68-212 mM potassium Pi' Fractions containing activity (60 ml eluting at 130-160 mMPi) were pooled, and the pool was concentrated to 15 ml in a Centriprep 30 concentrator (Amicon), The pool was diluted with H20 to 30 ml, and 5.2 g of solid ammonium sulfate was added over several minutes with stirring
Purification of Wild -typ e GSP Synthetase-Fractionation of E. coli B extracts by a sequence of streptomycin sulfate and ammonium sulfate precipitation steps followed by five chromatographic steps (OEAE-Sepharose, Mono-Q, hydroxya patite, phenyl-Su perose, and Mono-S) resulted in an estimated 2000 -5000-fold purification of GSP synthetase activity
Summary
General Materials-The ordered library of the E. coli genome [26] was kindly provided by Prof. All enzyme activity was found in the wash fractions, and these were pooled and loaded onto a 2.6 X 12-cm hydroxylapatite column equilibrated in 20 mM potassium phosphate buffer, pH 6.8 (buffer D), The column was developed with a 20-ml gradient of 20-68 mM potassium Pi' with a 300-ml gradient of212 mM potassium Pi' Fractions containing activity (60 ml eluting at 130-160 mMPi) were pooled, and the pool was concentrated to 15 ml in a Centriprep 30 concentrator (Amicon), The pool was diluted with H20 to 30 ml, and 5.2 g of solid ammonium sulfate was added over several minutes with stirring. Fractions containing activity (9 ml eluting at 0.64-0.60 M ammonium sulfate) were pooled and dialyzed against buffer C This solution was loaded onto a Mono-S HR 5/5 column, which was developed with a 2-mllinear gradient of 0-100 mM NaCI in buffer C followed by a 40-mllinear gradient of 100-500 mM NaCI in buffer C.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
