Expression, purification and characterization of glutamate cysteine ligase from Saccharomyces cerevisiae

Abstract

Glutathione is a critical cellular reductant and its homeostasis is required for maintenance of intracellular redox balance. glutamate‐cysteine ligase (GCL) catalyses the ATP‐dependent peptide bond formation between gamma‐carboxylate of glutamate and cysteine, the first step of glutathione biosynthesis. Mammalian GCL are heterodimers consisting of a catalytic subunit and a modifier subunit. The catalytic subunit catalyzes formation of glutamylcysteine and is inhibited by GSH, whereas the modifier subunit enhances the GCL affinity for L‐glutamate and decreases the inhibition by GSH. An examination of the S. cerevisiae genome revealed a single gene with sequence homology to the catalytic GCL subunit. The S. cerevisiae GCL catalytic subunit was cloned, expressed in E. coli and purified to homogeneity. Size exclusion chromatography and DLS techniques were used to examine the oligomeric state of GCL catalytic subunit. The kinetic activity of GCL catalytic subunit was measured using a coupled assay and apparent Km and Vmax values were determined. Kinetic constants were comparable to those reported for other eukaryotic GCL, and inhibition studies indicate that the enzyme is susceptible to competitive inhibition by the ultimate product of the pathway, glutathione. A proteomic method is currently being developed to identify the corresponding modifier subunit of S. cerevisiae GCL.