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

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 gamma-glutamylcysteine synthetase (gamma GCS), We have cloned and sequenced the 2037-base pair gene coding for the catalytic subunit of T, brucei gamma GCS, T, brucei gamma GCS appears to be encoded by a single copy gene, A transcript of about 2.3 kilobases was observed in procyclic trypomastigotes, The deduced amino acid sequence of 679 amino acids shares 45, 41, and 36% sequence identity with mammalian, Caenorhabditis elegans, and yeast gamma GCS, respectively, The T. brucei gamma GCS 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 gamma GCS, 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-alpha-aminobutyrate (apparent K-m = 10 mM) is a poor substitute for L-Cys in the T. brucei gamma GCS-catalyzed reaction, T. brucei gamma GCS 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 gamma GCS suggest that the substrate binding pocket and the mechanism of enzyme regulation differ from the mammalian enzyme, providing evidence that T, brucei gamma GCS could be a selective chemotherapeutic target for the treatment of trypanosomiasis.