Enhanced free radical generation of FALS-associated Cu,Zn-SOD mutants.

Familial amyotrophic lateral sclerosis (FALS) is an inherited disorder of motor neurons, which is associated with missense mutations in the Cu,Zn-superoxide dismutase (Cu,Zn-SOD) gene. Mice from the G93A transgenic line were reported to develop a syndrome of FALS. The fact that the symptoms occurred against a background of normal mouse Cu,Zn-SOD activity suggests that dominant, gain-of-function mutations in SOD play a role in the pathogenesis of FALS. We investigated the nature of this gain-of-function of FALS mutants. We have previously reported that Cu,Zn-SOD has the free radical-generating function in addition to normal dismutation activity. These two enzymic activities were compared by using mutants (G93A and A4V) and the wild-type Cu,Zn-SOD prepared by recombinant method. Our results showed that the wild-type, G93A, and A4V enzymes have identical dismutation activity. However, the free radical-generating function of the G93A and A4V mutants, as measured by the spin trapping and EPR method, is enhanced relative to that of the wild-type enzyme (wild type < G93A < A4V), particularly at lower H(2)O(2) concentrations. This is due to the decrease in the K(m) value for H(2)O(2), wild-type > G93A > A4V. The catalytic activity to generate free radicals is correlated to the clinical severity of the disorder induced by these mutant enzymes. Furthermore, we found that intact FALS mutants failed to enhance tyrosine nitration. Together our results indicate that the amyotrophic lateral sclerosis symptoms are not caused by the reduction of Cu,Zn-SOD dismutation activity with the mutant enzymes; rather, it is induced in part by enhancement of the free radical-generating function.