Expression, purification, and characterization of a familial amyotrophic lateral sclerosis-associated D90A Cu,Zn-superoxide dismutase mutant.

Cu,Zn-superoxide dismutase (SOD) is known to be a locus of mutation in familial amyotrophic lateral sclerosis (FALS). We cloned the FALS mutant, D90A, and wild-type of human Cu,Zn-SOD, overexpressed them in E. coli, purified the proteins, and studied their properties. We investigated their enzymic activities for catalyzing the dismutation of superoxide anions and the generation of free radicals with H2O2 as a substrate. Our results showed that both wild-type and mutant enzymes have identical dismutation activities. However, the hydroxyl radical-generating function of the D90A mutant, as measured using a 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonate), was enhanced relative to that of the wild-type enzyme. Catalysis of this reaction by D90A was more sensitive to inhibition by the copper chelators, penicillamine and diethyldithiocarbamate, than was catalysis by wild-type Cu,Zn-SOD. Our study suggests that this gain-of-function of FALS mutant may, in part, be responsible for the development of FALS symptoms.