Yang Yi, Chen Ying, Johansson Elisabet, Schneider Scott N, Shertzer Howard G, Nebert Daniel W, Dalton Timothy P
Department of Environmental Health and Center for Environmental Genetics, University of Cincinnati Medical Center, P.O. Box 670056, Cincinnati OH 45267-005, United States.
Biochem Pharmacol. 2007 Jul 15;74(2):372-81. doi: 10.1016/j.bcp.2007.02.003. Epub 2007 Feb 12.
Glutamate-cysteine ligase (GCL) is the rate-limiting enzyme in the glutathione (GSH) biosynthesis pathway. This enzyme is a heterodimer, comprising a catalytic subunit (GCLC) and a regulatory subunit (GCLM). Although GCLC alone can catalyze the formation of l-gamma-glutamyl-l-cysteine, its binding with GCLM enhances the enzyme activity by lowering the K(m) for glutamate and ATP, and increasing the K(i) for GSH inhibition. To characterize the enzyme structure-function relationship, we investigated the heterodimer formation between GCLC and GCLM, in vivo using the yeast two-hybrid system, and in vitro using affinity chromatography. A strong and specific interaction between GCLC and GCLM was observed in both systems. Deletion analysis indicated that most regions, except a portion of the C-terminal region of GCLC and a portion of the N-terminal region of GCLM, are required for the interaction to occur. Point mutations of selected amino acids were also tested for the binding activity. The GCLC Cys248Ala/Cys249Ala and Pro158Leu mutations enzyme showed the same strength of binding to GCLM as did wild-type GCLC, yet the catalytic activity was dramatically decreased. The results suggest that the heterodimer formation may not be dependent on primary amino-acid sequence but, instead, involves a complex formation of the tertiary structure of both proteins.
谷氨酸-半胱氨酸连接酶（GCL）是谷胱甘肽（GSH）生物合成途径中的限速酶。该酶是一种异二聚体，由催化亚基（GCLC）和调节亚基（GCLM）组成。尽管单独的GCLC可以催化L-γ-谷氨酰-L-半胱氨酸的形成，但其与GCLM的结合通过降低对谷氨酸和ATP的K（m）以及增加对GSH抑制的K（i）来增强酶活性。为了表征酶的结构-功能关系，我们在体内使用酵母双杂交系统，在体外使用亲和色谱法研究了GCLC和GCLM之间的异二聚体形成。在两个系统中均观察到GCLC和GCLM之间存在强烈且特异性的相互作用。缺失分析表明，除了GCLC C末端区域的一部分和GCLM N末端区域的一部分外，大多数区域对于相互作用的发生都是必需的。还测试了所选氨基酸的点突变的结合活性。GCLC Cys248Ala/Cys249Ala和Pro158Leu突变酶与野生型GCLC显示出与GCLM相同的结合强度，但催化活性显著降低。结果表明，异二聚体的形成可能不依赖于一级氨基酸序列，而是涉及两种蛋白质三级结构的复杂形成。
