Expression and characterization of human glutamate-cysteine ligase.
Glutamate-cysteine ligase (GLCL) catalyzes the rate-limiting step in glutathione biosynthesis. GLCL comprises regulatory (GLCLR) and catalytic (GLCLC) subunits. To understand better the structure-function relationship of GLCL subunits and holoenzyme, human GLCLR and GLCLC genes were inserted into the baculovirus genome. Recombinant hGLCLR andhGLCLC were produced in cells infected with recombinant baculoviruses, and homogeneous hGLCL subunits and holoenzyme were purified from cell lysates with a Ni-NTA resin. Purified recombinant hGLCL holoenzyme was catalytically more active than hGLCLC with L-glutamate, L-alpha-aminobutyrate, and ATP as substrates. The selectivity of purified hGLCL holoenzyme for L-glutamate, L-alpha-aminobutyrate, or L-cysteine was significantly higher than for hGLCLC. Glutathione was a noncompetitive inhibitor for both hGLCL holoenzyme and hGLCLC. hGLCLC was more sensitive to inhibition by glutathione than hGLCL holoenzyme. Deletion of the first 25 amino acid residues at the amino terminus of GLCLC dramatically decreased GLCL activity, indicating that the amino terminus of GLCLC is required for full catalytic activity. Expressed and purified hGLCL provides a useful tool to investigate glutathione biosynthesis in vitro.
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Department of Pharmacology and Physiology, University of Rochester, 601 Elmwood Avenue, Rochester, New York, 14642, USA.
Anders
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D000818:Animals; D016367:Baculoviridae; D002460:Cell Line; D003001:Cloning, Molecular; D005721:Glutamate-Cysteine Ligase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D007313:Insecta; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione