Identification of an important cysteine residue in human glutamate-cysteine ligase catalytic subunit by site-directed mutagenesis.
Identification of an important cysteine residue in human glutamate-cysteine ligase catalytic subunit by site-directed mutagenesis.
复制标题
通过定点诱变鉴定人谷氨酸-半胱氨酸连接酶催化亚基中重要的半胱氨酸残基。
DOI：
10.1042/bj3360675
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Anders,MW
中科院分区：
文献类型：
--
作者：
Tu,Z;Anders,MW
谷氨酸-半胱氨酸连接酶（GLCL）催化谷胱甘肽生物合成中的限速步骤。为了鉴定GLCL中参与其活性的半胱氨酸残基，通过基于PCR的定点突变将人GLCL催化亚基（hGLCLC）中的8个保守半胱氨酸残基替换为甘氨酸残基。重组hGLCLC和hGLCL全酶用杆状病毒表达系统表达和纯化。用突变体hGLCLC-C553 G纯化的hGLCL全酶的活性为110±12μmol/h/mg蛋白质，而野生型为370±20μmol/h/mg蛋白质。具有hGLCLC-C52 G、-C248 G、-C249 G、-C295 G、-C491 G、-C501 G或-C605 G的全酶显示与野生型相似的活性。含有hGLCLC-C553 G的hGLCL的Km值略低于野生型的Km值，表明在hGLCLC中用Gly替换半胱氨酸-553对酶的底物结合没有显著影响。hGLCLC-C553 G比野生型hGLCLC更容易从hGLC 1解离。将hGLCLC-C553 G与等摩尔量的纯化的hGLCL调节亚基（hGLCL）在室温下孵育30 min后，GLCL活性增加了11%，而将野生型hGLCL与hGLCL孵育10 min后，GLCL活性增加了110%。这些结果表明，hGLCLC中的半胱氨酸-553参与hGLCLC和hGLC 3之间的异二聚体形成。
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±12µmol/h per mg of protein compared with 370±20µmol/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. TheKmvalues 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.