Structure determination and interactions studies of the human gamma-glutamylcysteine synthetase light subunit by nuclear magnetic resonance

Glutathione is a ubiquitous tripeptide and the major cellular antioxidant found in most aerobic organisms. While glutathione deficiency leaves cells with an increased sensitivity to oxidative stress and contributes to the pathogenesis of a large range of diseases, high levels of glutathione have been shown to contribute to drug resistance in tumor cells and in some parasitic organisms. Biosynthesis of glutathione occurs in two highly conserved, ATP-dependent reactions. The first and rate-limiting reaction is catalyzed by γ-glutamylcysteine synthetase (γ-GCS; also known as glutamate cysteine ligase, or GCL). In higher eukaryotes, γ-GCS is a heterodimer composed of a heavy (∼73 kDa) subunit with all of the catalytic activity and a light (∼31 kDa) subunit with a regulatory function. The light subunit lowers the K m of the heavy subunit for the substrate glutamate and decreases the K i for inhibition by glutathione to biologically relevant concentration levels, and is thereby required for optimal γ-GCS activity in vivo . This dissertation presents a structural analysis of the human γ-GCS light subunit (γ-GCS L ). The solution structure for a core region of this protein has been determined to an RMSD of 1.2 Å by NMR spectroscopy. This structure exhibits a unique fold with some similarity to a TIM-barrel (αβ) 8 structure. Additional NMR experiments are described which are used to map the non-covalent binding surface of the heavy subunit onto the surface of γ-GCS L . These results suggest that γ-GCS heterodimer formation is directed by a weak electrostatic interaction. Prior the work presented in this dissertation, no structural information was available for either subunit of eukaryotic γ-GCS and the mechanism by which γ-GCS L influences the catalytic activity of the γ-GCS heterodimer was poorly understood. The results described here present a platform for further investigation of the eukaryotic γ-GCS holoenzyme on a structural level.