The IDP01828 glutamate-cysteine ligase from Francisella tularensis subsp. tularensis SCHU S4 is a rate-limiting enzyme that participates in the glutamate and glutathione metabolism and is a target for development of potential therapeutic agents against parasites and cancer. The catalytic mechanism of the enzyme has been proposed to involve the initial activation of the γ-carboxyl group of L-Glu by ATP-phosphorylation to form a γ-glutamylphosphate intermediate, followed by the nucleophilic attack of the amino group of L-Cys on the carbonyl to generate a tetrahedral transition state. Thus, the enzyme utilizes three substrates, ATP, L-Glu and L-Cys.
The IDP01828 protein was initially co-crystallized with the ATP analog, 1mM AMPPNP, in the presence of 1mM MgCl2, however, the final crystal structure (PDB ID code 3NZT) contains only AMP part of the ligand in the active site. The nucleoside part of AMP sits in a deep groove at a wider opening of a 29 A long negatively charged tunnel, which goes through the entire protein. Two longest beta strands (residues ranges 10-21 and 136-141) of the 47 % helical tangled ligase form a base of this depression. The phosphate group of the nucleotide points toward the positively charged pocket at the beginning of the wider opening of the tunnel. Four sulfate ions have been modeled inside the tunnel in the vicinity of where two other substrates, L-Glu and L-Cys, shall bind. The opposite end of the tunnel is half way closed with a Asp218-Lys227 salt bridge, which might also locally stabilize the tertiary structure of the protein.