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 mut ant, D90A, and wild-type of human Cu,Zn-SOD, overexpressed them in E. coli, purified the proteins, and studied their properties, We investig ated their enzymic activities for catalyzing the dismutation of supero xide anions and the generation of free radicals with H2O2 as a substra te, Our results showed that both wild-type and mutant enzymes have ide ntical dismutation activities. However, the hydroxyl radical-generatin g function of the D90A mutant, as measured using a 2,2'-azinobis-(3-et hylbenzthiazoline-6-sulfonate), was enhanced relative to that of the w ild-type enzyme. Catalysis of this reaction by D90A was more sensitive to inhibition by the copper chelators, penicillamine and diethyldithi ocarbamate, than was catalysis by wild-type Cu,Zn-SOD. Our study sugge sts that this gain-of-function of FALS mutant may, in part, be respons ible for the development of FALS symptoms.