Convenient Isolation and Kinetic Mechanism of Glutathionylspermidine Synthetase fromCrithidia fasciculata

Kerstin Koenig, Ulrich Menge, Michael Kiess, Victor Wray, Leopold Flohé
1997 Journal of Biological Chemistry  
Trypanothione, the essential metabolite in the oxidant defense system of trypanosomatids, is synthesized by two distinct proteins, glutathionylspermidine synthetase and trypanothione synthetase. Glutathionylspermidine synthetase was purified to homogeneity from the trypanosomatid Crithidia fasciculata by aqueous two-phase systems and chromatography. The enzyme showed a specific activity of 38 mol of glutathionylspermidine formed per min per mg of protein. Its molecular mass was 78 kDa in
more » ... yacrylamide gel electrophoresis, and it appeared predominantly monomeric in native polyacrylamide gel electrophoresis and gel filtration. The isoelectric point was at pH 4.6, and the pH optimum was near 7.6. Partial amino acid sequencing revealed homology with, but low similarity to, the glutathionylspermidine synthetase/amidase of Escherichia coli, and amidase activity was not detected in glutathionylspermidine synthetase of C. fasciculata. The kinetics of trypanosomatid glutathionylspermidine synthetase revealed a rapid equilibrium random mechanism with limiting K m values for Mg 2؉ -ATP, GSH, and spermidine of 0.25 ؎ 0.02, 2.51 ؎ 0.33, and 0.47 ؎ 0.09 mM, respectively, and a k cat of 415 ؎ 78 min ؊1 . Partial reactions at restricted cosubstrate supply were not detected by 31 P NMR, supporting the necessity of a quarternary complex formation for catalysis. ADP inhibited competitively with respect to ATP (K i ‫؍‬ 0.08 mM) and trypanothione exerted a feedback inhibition competitive with GSH (K i ‫؍‬ 0.48 mM). We suggest that subscribers photocopy these corrections and insert the photocopies at the appropriate places where the article to be corrected originally appeared. Authors are urged to introduce these corrections into any reprints they distribute. Secondary (abstract) services are urged to carry notice of these corrections as prominently as they carried the original abstracts.
doi:10.1074/jbc.272.18.11908 pmid:9115252 fatcat:vdjpsygjz5auxfhqnlfmfcdf7e