Convenient Isolation and Kinetic Mechanism of Glutathionylspermidine Synthetase from Crithidia fasciculata

Abstract

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 SDS-polyacrylamide 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.

Trypanothione Synthesis in Crithidia Revisited

“…Subtracting the k cat value of the TryS reaction (8.7 s Ϫ1 ; see below) from the overall k cat yields a real k cat for the GspS activity of 4.2 s Ϫ1 . The k cat for the GspS reaction of Cf-TryS is close to those reported for the "real" GspS of C. fasciculata (24,25) and E. coli (29). Cf-TryS, however, mark- edly differs from typical GspS enzymes in having a more than 10 times higher K m for spermidine, which explains that only at high spermidine concentrations the GspS activity starts to compete with the TryS activity (Fig.…”

“…There is not even a relevant difference in k cat for the TryS and GspS reactions of the three TryS species, and in absolute terms these values compare well with those reported for GspS-type enzymes (6.9 Ϫ15.5 s Ϫ1 for Cf-GspS (24, 25) and 7 s Ϫ1 for E. coli GspS (29)). Most interestingly, however, the K m values of Cf-TryS for spermidine are significantly higher than those for Gsp and those of Cf-GspS for spermidine (59-470 M (24,25)), whereas the TryS of trypanosomes, which seems to lack a GspS, display lower K m values for spermidine.…”

“…Heterologous expression and purification of mutants was performed as described above for the wildtype reCf-TryS. Substrate Specificity of Wild-type or Mutant reCf-TryS-Specificity was investigated by HPLC analysis of products (24). Enzymatic activities of recombinant proteins (1-10 g of protein for wild-type sequences or 0.05-1 mg of protein for mutant proteins) were incubated at 25°C in a volume of 0.9 ml containing 50 mM bis-Tris-propane, 50 mM Tris, pH 7.5, 5 mM Mg 2ϩ -ATP, 2-5 mM GSH, and 1-10 mM spermidine, 1-10 mM N 8 -acetylspermidine, or 2 mM Gsp, depending on the substrate specificity to be determined.…”

“…The thiols were reacted with monobrobimane and methansulfonic acid at different time points and stored for 24 h at RT before application to a 92 Nucleosil 300 -5C8 HPLC column. Bimane derivatives of thiols were detected fluorometrically as described (24). GSH, Gsp, and T(SH) 2 were used for calibration (a differentiation between N 1 -and N 8 -glutathionylspermidine was not achieved).…”

Thiol Metabolism of the Trypanosomatids as Potential Drug Targets

“…These activities were originally attibuted to a single protein (9), but the two activities from Crithidia fasciculata were recently separated by hydrophobic interaction chromatography (10). In a separate study glutathionylspermidine synthase was also isolated from Crithidia fasciculata (11). Unfortunately, active enzymes could not be recovered following heterologous expression of the Crithidia fasciculata enzymes in E. coli or in yeast cells (10).…”