期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 294, 期 9, 页码 3235-3248出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.RA118.006366
关键词
trypanosome; glutathionylation; oxidation-reduction (redox); enzyme catalysis; disulfide; thiol; fluorescence; trypanothione
资金
- Comision Sectorial de Investigacion Cientifica Universidad de la Republica [C601-348]
- International Centre for Genetic Engineering and Biotechnology [CRP/URU14-01]
- PRAT Universita degli Studi di Padova [COF 03/11, CPDA137397/13]
- European Comission [261863]
- Fondo para la Convergencia Estructural del Mercosur
Trypanosomes are flagellated protozoan parasites (kinetoplastids) that have a unique redox metabolism based on the small dithiol trypanothione (T(SH)(2)). Although GSH may still play a biological role in trypanosomatid parasites beyond being a building block of T(SH)(2), most of its functions are replaced by T(SH)(2) in these organisms. Consequently, trypanosomes have several enzymes adapted to using T(SH)(2) instead of GSH, including the glutaredoxins (Grxs). However, the mechanistic basis of Grx specificity for T(SH)(2) is unknown. Here, we combined fast-kinetic and biophysical approaches, including NMR, MS, and fluorescent tagging, to study the redox function of Grx1, the only cytosolic redox-active Grx in trypanosomes. We observed that Grx1 reduces GSH-containing disulfides (including oxidized trypanothione) in very fast reactions (k > 5 x 10(5) m(-1) s(-1)). We also noted that disulfides without a GSH are much slower oxidants, suggesting a strongly selective binding of the GSH molecule. Not surprisingly, oxidized Grx1 was also reduced very fast by T(SH)(2) (4.8 x 10(6) m(-1) s(-1)); however, GSH-mediated reduction was extremely slow (39 m(-1) s(-1)). This kinetic selectivity in the reduction step of the catalytic cycle suggests that Grx1 uses preferentially a dithiol mechanism, forming a disulfide on the active site during the oxidative half of the catalytic cycle and then being rapidly reduced by T(SH)(2) in the reductive half. Thus, the reduction of glutathionylated substrates avoids GSSG accumulation in an organism lacking GSH reductase. These findings suggest that Grx1 has played an important adaptive role during the rewiring of the thiol-redox metabolism of kinetoplastids.
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