Catalytic and regulatory properties of the heavy subunit of rat kidney gamma-glutamylcysteine synthetase.
Catalytic and regulatory properties of the heavy subunit of rat kidney gamma-glutamylcysteine synthetase.
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DOI：
10.1016/s0021-9258(19)36569-x
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发表时间：
1993-09
期刊：
影响因子：
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通讯作者：
C. Huang;Long-Sen Chang;Mary E. Anderson;A. Meister
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文献类型：
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作者：
C. Huang;Long-Sen Chang;Mary E. Anderson;A. Meister
γ-谷氨酰半胱氨酸合成酶(大鼠肾脏)催化GSH合成的第一步，经二硫苏糖醇(DTT)处理后，可通过天然凝胶电泳法分离为亚基(M(R)73,000和27,700)；重亚单位显示出催化活性和GSH的反馈抑制(Seelig，G.F.，Simondsen，R.P.和Meister，A.(1984)J.Biol)。化学。259,9345-9347)，并对其进行了克隆和测序。化学。265、1588-1593)。在此，我们在大肠杆菌中表达了重亚单位的基因，并从大肠杆菌γ-谷氨酰半胱氨酸合成酶中分离纯化了重组酶。与全酶相比，重组酶和分离的重亚单位对谷氨酸的亲和力低得多，而对GSH抑制的敏感性更高，这表明单独的重亚单位在体内不是很活跃。GSH类似物，γ-谷氨酸-α-氨基丁酰甘氨酸(眼酸)，仅有轻微的抑制作用，但在用DTT处理全酶后抑制更多。相反，在没有DTT处理的情况下，邻苯二甲酸基本上抑制重组和分离的重亚单位酶。我们的结论是：(A)LIGH亚基对谷氨酸和GSH的亲和力有调节作用，(B)GSH的反馈抑制涉及酶的还原以及GSH和谷氨酸对谷氨酸位置的竞争。
gamma-Glutamylcysteine synthetase (rat kidney), which catalyzes the first step of GSH synthesis, can be dissociated into subunits (M(r) 73,000 and 27,700) by native gel electrophoresis after treatment with dithiothreitol (DTT); the heavy subunit, which exhibits catalytic activity and feedback inhibition by GSH (Seelig, G. F., Simondsen, R. P., and Meister, A. (1984) J. Biol. Chem. 259, 9345-9347), was cloned and sequenced (Yan, N., and Meister, A. (1990) J. Biol. Chem. 265, 1588-1593). Here, the cDNA for the heavy sub unit was expressed in Escherichia coli, and the recombinant enzyme was separated from E. coli gamma-glutamylcysteine synthetase and purified. The recombinant enzyme and the isolated heavy subunit have much lower affinity for glutamate and higher sensitivity to GSH inhibition than the holoenzyme, suggesting that the heavy subunit alone would not be very active in vivo. A GSH analog, gamma-Glu-alpha-aminobutyryl-Gly (ophthalmic acid), inhibits only slightly, but inhibits much more after treatment of the holoenzyme with DTT. In contrast, ophthalmic acid inhibits the recombinant and isolated heavy subunit enzymes substantially without DTT treatment. We conclude that (a) the light subunit has a regulatory function affecting the affinity of the enzyme for glutamate and GSH and (b) feedback inhibition by GSH involves reduction of the enzyme and also competition between GSH and glutamate for the glutamate site.