Interaction between the catalytic and modifier subunits of glutamate-cysteine ligase.
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.
Yang
Yi
Y
Department of Environmental Health and Center for Environmental Genetics, University of Cincinnati Medical Center, P.O. Box 670056, Cincinnati OH 45267-005, United States.
Chen
Ying
Y
Johansson
Elisabet
E
Schneider
Scott N
SN
Shertzer
Howard G
HG
Nebert
Daniel W
DW
Dalton
Timothy P
TP
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
D021122:Protein Subunits; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione