Characterization of Trypanosoma brucei γ-glutamylcysteine synthetase, an essential enzyme in the biosynthesis of trypanothione (diglutathionylspermidine)

Deirdre V. Lueder, Margaret A. Phillips

Research output: Contribution to journalArticle

76 Scopus citations

Abstract

The parasitic protozoan Trypanosoma brucei maintains redox balance by synthesizing a conjugate of glutathione and spermidine termed trypanothione. The first committed step in the biosynthesis of glutathione, and thereby trypanothione, is catalyzed by the enzyme γ-glutamylcysteine synthetase (γGCS). We have cloned and sequenced the 2037-base pair gene coding for the catalytic subunit of T. brucei γGCS. T. brucei γGCS appears to be encoded by a single copy gene. A transcript of about 2.3 kilobases was observed in procyclic trypomastigotes. The deduced amine acid sequence of 679 amine acids shares 45, 41, and 36% sequence identity with mammalian, Caenorhabditis elegans, and yeast γGCS, respectively. The T. brucei γGCS gene was expressed in E. coli; the purified 77.4-kDa enzyme catalyzes the ligation of L-Glu to L-Cys with a k(cat) of 10 s-1, confirming that the gene encodes the functional catalytic subunit of γGCS. The apparent K(m) values measured for the three natural substrates L-Glu, L-Cys, and ATP are 0.24, 0.69, and 0.07 mM, respectively. Unlike the mammalian enzyme, L-α-aminobutyrate (apparent K(m) = 10 mM) is a poor substitute for L-Cys in the T. brucei γGCS-catalyzed reaction. T. brucei γGCS is feedback-inhibited by glutathione (apparent K(I) = 1.1 mM), and it is inactivated by cystamine and buthionine sulfoximine. The kinetic properties of recombinant T. brucei γGCS suggest that the substrate binding pocket and the mechanism of enzyme regulation differ from the mammalian enzyme, providing evidence that T. brucei γGCS could be a selective chemotherapeutic target for the treatment of trypanosomiasis.

Original languageEnglish (US)
Pages (from-to)17485-17490
Number of pages6
JournalJournal of Biological Chemistry
Volume271
Issue number29
DOIs
StatePublished - 1996

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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