Identification of an important cysteine residue in human glutamate-cysteine ligase catalytic subunit by site-directed mutagenesis.
Glutamate-cysteine ligase (GLCL) catalyses the rate-limiting step in glutathione biosynthesis. To identify cysteine residues in GLCL that are involved in its activity, eight conserved cysteine residues in human GLCL catalytic subunit (hGLCLC) were replaced with glycine residues by PCR-based site-directed mutagenesis. Both recombinant hGLCLC and hGLCL holoenzyme were expressed and purified with a baculovirus expression system. The activity of purified hGLCL holoenzyme with the mutant hGLCLC-C553G was 110+/-12 micromol/h per mg of protein compared with 370+/-20 micromol/h per mg of protein for the wild-type. Holoenzymes with hGLCLC-C52G, -C248G, -C249G, -C295G, -C491G, -C501G or -C605G showed activities similar to the wild type. The Km values of hGLCL containing hGLCLC-C553G were slightly lower than those of the wild type, indicating that the replacement of cysteine-553 with Gly in hGLCLC did not significantly affect substrate binding by the enzyme. hGLCLC-C553G was more easily dissociated from hGLCLR than the wild-type hGLCLC. GLCL activity increased by 11% after hGLCLC-C553G was incubated with an equimolar amount of purified hGLCL regulatory subunit (hGLCLR) at room temperature for 30 min, but increased by 110% after wild-type hGLCLC was incubated with hGLCLR for 10 min. These results indicate that cysteine-553 in hGLCLC is involved in heterodimer formation between hGLCLC and hGLCLR.
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Department of Pharmacology and Physiology, School of Medicine and Dentistry, University of Rochester, 610 Elmwood Avenue, Box 711, Rochester, NY 14642, USA.
Anders
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MW
D019943:Amino Acid Substitution; D000818:Animals; D020134:Catalytic Domain / Q000235:genetics; D002460:Cell Line; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D019281:Dimerization; D004789:Enzyme Activation; D005721:Glutamate-Cysteine Ligase / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D018411:Spodoptera; D014162:Transfection
D005721:Glutamate-Cysteine Ligase; D003545:Cysteine