A FAMILIAL AMYOTROPHIC LATERAL SCLEROSIS-ASSOCIATED A4V CU,ZN-SUPEROXIDE DISMUTASE MUTANT HAS A LOWER K-M FOR HYDROGEN-PEROXIDE

Point mutations of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) have been li nked to familial amyotrophic lateral sclerosis (FALS). We reported tha t Cu,Zn-SOD can catalyze free radical generation and a FALS mutant, G9 3A, exhibits an enhanced free radical-generating activity, while its d ismutation activity is identical to that of the wild-type enzyme (Yim, M. B., Bang J.-H., Yim, H.-S., Kwak, H.-S., Chock, P. B., and Stadtma n, E. R. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5709-5714). The A4 V 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-Yase k, 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. (1 994) Rum. Mol. Genet. 3, 981-987). We cloned the cDNA for the FALS A4V mutant, overexpressed the protein in Sf9 insect cells, purified the p rotein, and studied its enzymic activities. Our results show that the mutant and wild-type enzymes contain one copper ion per subunit and ha ve identical dismutation activities. However, the free radical-generat ing 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 K-m value for H2O2, wild-type > G93A > A4V, while the k(cat) i s identical for these enzymes. Thus, the FALS symptoms are not associa ted with the reduction in the dismutation activity of the mutant enzym e. The fact that the A4V mutant has the lowest K-m for H2O2 is correla ted to the clinical severity observed with the A4V patients, if FALS i s associated with a differential gain of the free radical-generating f unction of the Cu,Zn-SOD mutant.