Title: Interaction between the catalytic and modifier subunits of glutamate-cysteine ligase.

Authors: Yang, Yi; Chen, Ying; Johansson, Elisabet; Schneider, Scott N; Shertzer, Howard G; Nebert, Daniel W; Dalton, Timothy P

Published In Biochem Pharmacol, (2007 Jul 15)

Abstract: Glutamate-cysteine ligase (GCL) is the rate-limiting enzyme in the glutathione (GSH) biosynthesis pathway. This enzyme is a heterodimer, comprising a catalytic subunit (GCLC) and a regulatory subunit (GCLM). Although GCLC alone can catalyze the formation of l-gamma-glutamyl-l-cysteine, its binding with GCLM enhances the enzyme activity by lowering the K(m) for glutamate and ATP, and increasing the K(i) for GSH inhibition. To characterize the enzyme structure-function relationship, we investigated the heterodimer formation between GCLC and GCLM, in vivo using the yeast two-hybrid system, and in vitro using affinity chromatography. A strong and specific interaction between GCLC and GCLM was observed in both systems. Deletion analysis indicated that most regions, except a portion of the C-terminal region of GCLC and a portion of the N-terminal region of GCLM, are required for the interaction to occur. Point mutations of selected amino acids were also tested for the binding activity. The GCLC Cys248Ala/Cys249Ala and Pro158Leu mutations enzyme showed the same strength of binding to GCLM as did wild-type GCLC, yet the catalytic activity was dramatically decreased. The results suggest that the heterodimer formation may not be dependent on primary amino-acid sequence but, instead, involves a complex formation of the tertiary structure of both proteins.

PubMed ID: 17517378

MeSH Terms: Catalysis; Catalytic Domain; Dimerization; Glutamate-Cysteine Ligase/chemistry*; Glutamate-Cysteine Ligase/metabolism; Glutathione/biosynthesis; Protein Subunits; Saccharomyces cerevisiae/genetics; Structure-Activity Relationship; Two-Hybrid System Techniques