title: Interaction between the catalytic and modifier subunits of glutamate-cysteine ligase.
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.
authors.affiliation: Department of Environmental Health and Center for Environmental Genetics, University of Cincinnati Medical Center, P.O. Box 670056, Cincinnati OH 45267-005, United States.
authors.forename: Yi
authors.initials: Y
authors.lastname: Yang
authors.forename: Ying
authors.initials: Y
authors.lastname: Chen
authors.forename: Elisabet
authors.initials: E
authors.lastname: Johansson
authors.forename: Scott N
authors.initials: SN
authors.lastname: Schneider
authors.forename: Howard G
authors.initials: HG
authors.lastname: Shertzer
authors.forename: Daniel W
authors.initials: DW
authors.lastname: Nebert
authors.forename: Timothy P
authors.initials: TP
authors.lastname: Dalton
mesh_terms: D002384:Catalysis; D020134:Catalytic Domain; D019281:Dimerization; D005721:Glutamate-Cysteine Ligase / Q000737:chemistry* / Q000378:metabolism; D005978:Glutathione / Q000096:biosynthesis; D021122:Protein Subunits; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013329:Structure-Activity Relationship; D020798:Two-Hybrid System Techniques
chemical_list: D021122:Protein Subunits; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione