GSH is the major low-molecular-mass thiol in most organisms. The tripeptide maintains a reduced intracellular environment and protects cellular components from damaging oxidation. GSH is synthesized by the action of two ATP-dependent enzymic steps, in which γ-glutamylcysteine synthetase (γ-GCS) catalyses the ligation of glutamate and cysteine and subsequently glutathione synthetase (GS) adds glycine to the dipeptide. Recently it was shown that the synthesis of γ-glutamylcysteine is crucial for the survival of the erythrocytic stages of the malaria parasite Plasmodium falciparum by using the specific γ-GCS inhibitor buthionine sulphoximine. In order to investigate further the synthetic pathway of the tripeptide in the parasite, GS was cloned and expressed recombinantly. The deduced amino acid sequence of P. falciparum GS shares only a moderate degree of identity with other known GSs, but the residues responsible for substrate and co-factor binding are almost all conserved, with the exception of the ones involved in γ-glutamylcysteine binding. The protein is active as a dimer, with a subunit molecular mass of 77kDa, and the addition of reducing reagents such as dithiothreitol is essential in maintaining enzymic activity, indicating that thiol groups are important for stability and enzymic activity. The Kappm values for γ-glutamyl-α-aminobutyrate, ATP and glycine were determined to be 107.1μM, 59.1μM and 5.04mM, respectively, and the Vmax of 5.24±0.7μmol·min−1·mg−1 was in the same range as that of the mammalian enzymes. However, the negative co-operativity observed for γ-glutamylcysteine binding to the rat enzyme was not found for the parasite protein. This may be due to the alteration of several amino acids in the γ-glutamylcysteine-binding site.

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