Substrate binding determinants of Trypanosoma brucei gamma-glutamylcysteine synthetase.
gamma-Glutamylcysteine synthetase (gamma-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 gamma-GCS is a structural homologue of glutamine synthetase (GS), providing the basis to build a model for the gamma-GCS active site [Abbott et al. (2001) J. Biol. Chem. 276, 42099-42107]. Substrate binding determinants in the active site of gamma-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 K(d) 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 gamma-aminobutyric acid, suggesting that R366 interacts with the alpha-carboxylate of L-Glu. Mutation of R491 to Ala decreased k(cat) for ATP hydrolysis by 70-fold; however, dipeptide product was only formed in 5% of these turnovers. These data suggest that R491 stabilizes the phosphorylated gamma-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 K(m) for ATP by 20-100-fold while having only modest effects on k(cat) or the apparent K(m)'s for the other substrates. Finally, mutation of R179, a conserved residue that is present in gamma-GCS, but not in GS, increased the apparent K(m) for both L-Cys and L-Glu.
Abbott
Jared J
JJ
Department of Pharmacology, The University of Texas Southwestern Medical Center at Dallas, 75390-9041, USA.
Ford
Jennifer L
JL
Phillips
Margaret A
MA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D017931:DNA Primers; D005721:Glutamate-Cysteine Ligase / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity; D014346:Trypanosoma brucei brucei / Q000201:enzymology*; D014461:Ultracentrifugation
D017931:DNA Primers; D005721:Glutamate-Cysteine Ligase