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 glutat hione 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-dir ected mutagenesis. Both recombinant hGLCLC and hGLCL holoenzyme were expres sed and purified with a baculovirus expression system. The activity of puri fied hGLCL holoenzyme with the mutant hGLCLC-C553G was 110 +/- 12 mu mol/h per mg of protein compared with 370 +/- 20 mu mol/h per mg of protein for t he wild-type. Holoenzymes with hGLCLC-C52G, -C248G, -C249G, -C295G, -C491G, -C501G or -C605G showed activities similar to the wild type. The K-m, valu es of hGLCL containing hGLCLC-C553G were slightly lower than those of the w ild type, indicating that the replacement of cysteine-553 with Gly in hGLCL C did not significantly affect substrate binding by the enzyme. hGLCLC-C553 G was more easily dissociated from hGLCLR than the wild-type hGLCLC. GLCL a ctivity increased by 11% after hGLCLC-C553G was incubated with an equimolar amount of purified hGLCL regulatory subunit (hGLCLR) at room temperature f or 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 i nvolved in heterodimer formation between hGLCLC and hGLCLR.