Yim H S, Kang J H, Chock P B, Stadtman E R, Yim M B
Laboratory of Biochemistry, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Biol Chem. 1997 Apr 4;272(14):8861-3. doi: 10.1074/jbc.272.14.8861.
Point mutations of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) have been linked to familial amyotrophic lateral sclerosis (FALS). We reported that Cu,Zn-SOD can catalyze free radical generation and a FALS mutant, G93A, exhibits an enhanced free radical-generating activity, while its dismutation activity is identical to that of the wild-type enzyme (Yim, M. B., Kang, J.-H., Yim, H.-S., Kwak, H.-S., Chock, P. B., and Stadtman, E. R. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5709-5714). The A4V mutation is both the most commonly detected of FALS-associated SOD1 mutations and among the most clinically severe (Rosen, D. R., Bowling, A. C., Patterson, D., Usdin, T. B., Sapp, P., Mezey, E., McKenna-Yasek, D., O'Regan, J. P., Rahmani, Z., Ferrante, R. J., Brownstein, M. J., Kowall, N. W., Beal, M. F., Horvitz, H. R., and Brown, R. H., Jr. (1994) Hum. Mol. Genet. 3, 981-987). We cloned the cDNA for the FALS A4V mutant, overexpressed the protein in Sf9 insect cells, purified the protein, and studied its enzymic activities. Our results show that the mutant and wild-type enzymes contain one copper ion per subunit and have identical dismutation activities. However, the free radical-generating activity of the mutant, as measured by the spin trapping method at low H2O2 concentration, is enhanced relative to that of the wild-type and G93A enzyme (wild-type < G93A < A4V). This is due to the decrease in the Km value for H2O2, wild-type > G93A > A4V, while the kcat is identical for these enzymes. Thus, the FALS symptoms are not associated with the reduction in the dismutation activity of the mutant enzyme. The fact that the A4V mutant has the lowest Km for H2O2 is correlated to the clinical severity observed with the A4V patients, if FALS is associated with a differential gain of the free radical-generating function of the Cu,Zn-SOD mutant.
铜锌超氧化物歧化酶（Cu,Zn-SOD）的点突变与家族性肌萎缩侧索硬化症（FALS）有关。我们报道过Cu,Zn-SOD能催化自由基生成，一种FALS突变体G93A表现出增强的自由基生成活性，而其歧化活性与野生型酶相同（Yim, M. B., Kang, J.-H., Yim, H.-S., Kwak, H.-S., Chock, P. B., and Stadtman, E. R. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5709 - 5714）。A4V突变是FALS相关SOD1突变中最常检测到的，也是临床症状最严重的突变之一（Rosen, D. R., Bowling, A. C., Patterson, D., Usdin, T. B., Sapp, P., Mezey, E., McKenna-Yasek, D., O'Regan, J. P., Rahmani, Z., Ferrante, R. J., Brownstein, M. J., Kowall, N. W., Beal, M. F., Horvitz, H. R., and Brown, R. H., Jr. (1994) Hum. Mol. Genet. 3, 981 - 987）。我们克隆了FALS A4V突变体的cDNA，在Sf9昆虫细胞中过量表达该蛋白，纯化了该蛋白，并研究了其酶活性。我们的结果表明，突变体酶和野生型酶每个亚基含有一个铜离子，并且具有相同的歧化活性。然而，通过自旋捕获法在低H2O2浓度下测量，突变体的自由基生成活性相对于野生型和G93A酶有所增强（野生型 < G93A < A4V）。这是由于H2O2的Km值降低，野生型 > G93A > A4V，而这些酶的kcat相同。因此，FALS症状与突变体酶歧化活性的降低无关。如果FALS与Cu,Zn-SOD突变体自由基生成功能的差异增强有关，那么A4V突变体对H2O2的Km值最低这一事实与A4V患者所观察到的临床严重程度相关。
