GLUTATHIONYLSPERMIDINE METABOLISM IN ESCHERICHIA-COLI - PURIFICATION,CLONING, OVERPRODUCTION, AND CHARACTERIZATION OF A BIFUNCTIONAL GLUTATHIONYLSPERMIDINE SYNTHETASE AMIDASE/

Glutathionylspermidine (GSP) synthetases of Trypanosomatidae and Esche richia coli couple hydrolysis of ATP (to ADP and P-i) with formation o f an amide bond between spermidine (N-(3-aminopropyl)-1,4-diamino-buta ne) 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 (N-1 ,N-8-bis-(glutathionyl)spermidine), and is a target for drug design. I n this study, GSP synthetase was purified to near homogeneity from E. coli B, the gene encoding it was isolated and sequenced, the enzyme wa s overexpressed and purified in quantity, and the recombinant enzyme w as characterized. The 70-kDa protein was found to have an unexpected s econd catalytic activity, glutathionylspermidine amide bond hydrolysis . Thus, the bifunctional GSP synthetase/amidase catalyzes opposing ami de bond-forming and -cleaving reactions, with net hydrolysis of ATP. T he synthetase activity is selectively abrogated by proteolytic cleavag e 81 residues from the C terminus, suggesting that the two activities reside in distinct domains (N-terminal amidase and C-terminal syntheta se). Proteolysis at this site is facile in the absence of substrates, but is inhibited in the presence of ATP, glutathione, and Mg2+. A seri es of analogs was used to probe the spermidine-binding site of the syn thetase activity. The activity of diaminopropane as a substrate, inact ivity of the C-4-C-8 diaminoalkanes, and greater loss of specificity f or analogs modified in the 3-aminopropyl moiety than for those modifie d in the 4-aminobutyl moiety indicate that the enzyme recognizes predo minantly 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 K-m and k(cat) for a set of difluoro-substituted spermidine derivatives sugges t that the enzyme may bind the minor, deprotonated form of the amine n ucleophile.