Furukawa Yoshiaki, Torres Andrew S, O'Halloran Thomas V
Department of Chemistry, Northwestern University, Evanston, IL 60208, USA.
EMBO J. 2004 Jul 21;23(14):2872-81. doi: 10.1038/sj.emboj.7600276. Epub 2004 Jun 24.
The antioxidant enzyme Cu,Zn-superoxide dismutase (SOD1) has the distinction of being one of the most abundant disulfide-containing protein known in the eukaryotic cytosol; however, neither catalytic nor physiological roles for the conserved disulfide are known. Here we show that the disulfide status of Saccharomyces cerevisiae SOD1 significantly affects the monomer-dimer equilibrium, the interaction with the copper chaperone CCS, and the activity of the enzyme itself. Disulfide formation in SOD1 by O2 is slow but is greatly accelerated by the Cu-bound form of CCS (Cu-CCS) in vivo and in vitro even in the presence of excess reductants; once formed, this disulfide is kinetically stable. Biochemical assays reveal that Cu-CCS facilitates Cys oxidation and disulfide isomerization in the stepwise conversion of the immature form of the enzyme to the active state. The immature form of SOD1 is most susceptible to oxidative insult and to aggregation reminiscent of that observed in amyotrophic lateral sclerosis. Thus Cu-CCS mediation of correct disulfide formation in SOD1 is important for regulation of enzyme activity and for prevention of misfolding or aggregation.
抗氧化酶铜锌超氧化物歧化酶（SOD1）是真核细胞胞质溶胶中已知的最丰富的含二硫键蛋白质之一；然而，保守二硫键的催化作用和生理作用均不为人所知。在此我们表明，酿酒酵母SOD1的二硫键状态显著影响单体 - 二聚体平衡、与铜伴侣蛋白CCS的相互作用以及酶本身的活性。在体内和体外，即使存在过量还原剂，O2在SOD1中形成二硫键的过程也很缓慢，但与铜结合的CCS形式（Cu - CCS）能极大地加速这一过程；一旦形成，这种二硫键在动力学上是稳定的。生化分析表明，在酶从未成熟形式逐步转变为活性状态的过程中，Cu - CCS促进半胱氨酸氧化和二硫键异构化。SOD1的未成熟形式最易受到氧化损伤和聚集，这与肌萎缩侧索硬化中观察到的情况类似。因此，Cu - CCS介导SOD1中正确二硫键的形成对于调节酶活性以及防止错误折叠或聚集很重要。
