Characterization of the Oligomeric Transition of E. coli γ‐Glutamylcysteine Ligase

Abstract

Gamma‐glutamylcysteine ligase (γ‐GCL) is the enzyme that catalyzes the rate‐limiting step in the synthesis of glutathione. Glutathione is necessary for the survival of most organisms, as it functions to detoxify cells. In vitro, purified E. coli γ‐GCL transitions from a monomeric (47kDa) to a dimeric (88kDa) form over time. In order to study whether oxidation and reduction chemistry is important in the transition from monomer to dimer, native polyacrylamide gel electrophoresis was used to monitor transitions between each form. Transitions were induced through the addition of the reducing agents Tris(2‐carboxyethyl)phosphine (TCEP) and dithiothreitol (DTT), and the oxidizing agent ascorbic acid. The addition of DTT and TCEP results in the conversion from dimer to monomer in a concentration dependent manner; however, differences in migration of these induced oligomers suggests there are shape differences between the native and induced forms. A transition to a higher order oligomer is observed upon the addition of the oxidizing agent ascorbic acid. The characterization of each oligomer and the oligomerization process will be important to advance our understanding of this enzyme system and will inform in vivo and in vitro studies with E. coli γ‐GCL.