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 micromol 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. The kinetics of trypanosomatid glutathionylspermidine synthetase revealed a rapid equilibrium random mechanism with limiting Km values for Mg2+-ATP, GSH, and spermidine of 0.25 +/- 0.02, 2.51 +/- 0.33, and 0.47 +/- 0. 09 mM, respectively, and a kcat of 415 +/- 78 min-1. Partial reactions at restricted cosubstrate supply were not detected by 31P NMR, supporting the necessity of a quarternary complex formation for catalysis. ADP inhibited competitively with respect to ATP (Ki = 0. 08 mM) and trypanothione exerted a feedback inhibition competitive with GSH (Ki = 0.48 mM).
Highlights
Glutathionylspermidine synthetase (GspS)1 catalyzes the first of two steps of trypanothione biosynthesis, the synthesis of glutathionylspermidine (Gsp) from GSH and spermidine with the consumption of ATP (1)
The precursor of TSH, Gsp, may have a distinct biological role. It was first identified in Escherichia coli (20), where it remains unprocessed to TSH due to the apparent lack of TSH synthetase
The optimized procedure was based on a factorial design of phase compositions (31), i.e. PEG6000/phosphate (7.5/13% (w/ w)), PEG4000/phosphate (8/14% (w/w)), PEG1550/phosphate
Summary
GspS, glutathionylspermidine synthetase; Gsp, glutathionylspermidine; TSH, trypanothione; TS, trypanothione synthetase; DTT, dithiothreitol; bis-Tris propane, 1,3-bis[tris(hydroxymethy)methylamino]propane; PEG, polyethylene glycol; PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liquid chromatography. TSH substitutes for GSH in the defense against hydroperoxides and derived reactive oxygen species because of its ability to reduce peroxides either enzymatically (7–9) or spontaneously (10) It thereby protects the parasitic trypanosomatids, which apparently are deficient in catalase and glutathione peroxidases (11), against oxidative stress for instance during host-defense reactions (9, 12, 13). In C. fasciculata, Gsp increases substantially during the transition from growth phase to stationary phase, while TSH simultaneously drops (22). These fluctuations of GSH conjugates or the associated variations in cellular spermidine levels have tentatively been implicated in growth regulation (2, 20, 21).
Sign-in/Register to view highlights & summary 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 callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
