Substrate binding determinants of Trypanosoma brucei γ-glutamylcysteine synthetase

Jared J. Abbott, Jennifer L. Ford, Margaret A. Phillips

Research output: Contribution to journalArticle

15 Scopus citations

Abstract

γ-Glutamylcysteine synthetase (γ-GCS) catalyzes the ATP-dependent ligation of L-Glu and L-Cys, which is the first step in de novo biosynthesis of the tripeptide glutathione. Recently it was demonstrated that γ-GCS is a structural homologue of glutamine synthetase (GS), providing the basis to build a model for the γ-GCS active site [Abbott et al. (201) J. Biol. Chem. 276, 42099-42107]. Substrate binding determinants in the active site of γ-GCS have been identified and characterized in the enzyme from the parasitic protozoa Trypanosoma brucei using this model as a guide for site-directed mutagenesis. R366 and R491 were identified as key determinants of L-Glu binding. Mutation of R366 to Ala increases the Kd for L-Glu by 160-fold and eliminates the positive cooperativity observed for the binding of L-Glu and ATP to the wild-type enzyme, based on a rapid equilibrium random mechanism of substrate binding. Unlike the wild-type enzyme, the R366A mutant enzyme was able to form product using the substrate analogue γ-aminobutyric acid, suggesting that R366 interacts with the α-carboxylate of L-Glu. Mutation of R491 to Ala decreased kcat for ATP hydrolysis by 70-fold; however, dipeptide product was only formed in 5% of these turnovers. These data suggest that R491 stabilizes the phosophorylated γ-carboxylate of L-Glu during nucleophilic attack by the L-Cys to form the dipeptide product. T323, R474, and R487 were predicted to be ATP binding determinants. Mutation of each of these residues to Ala increased the apparent Km for ATP by 20-100-fold while having only modest effects on kcat or the apparent Km's for the other substrates. Finally, mutation of R179, a conserved residue that is present in γ-GCS, but not in GS, increased the apparent Km for both L-Cys and L-Glu.

Original languageEnglish (US)
Pages (from-to)2741-2750
Number of pages10
JournalBiochemistry
Volume41
Issue number8
DOIs
StatePublished - Feb 26 2002

ASJC Scopus subject areas

  • Biochemistry

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