A gain-of-function of an amyotrophic lateral sclerosis-associated Cu,Zn-superoxide dismutase mutant: An enhancement of free radical formation due to a decrease in Km for hydrogen peroxide.

Abstract

Cu,Zn-superoxide dismutase (SOD) is known to be a locus of mutation in familial amyotrophic lateral sclerosis (FALS). Transgenic mice that express a mutant Cu,Zn-SOD, Because the levels of total Cu,Zn-SOD activity in these transgenic mice are greater than normal, the plausible interpretation of the ALS symptoms in these mice is that the mutant is in some way cytotoxic. The nature of this cytotoxic gain-offunction of the FALS mutants is yet to be identified.In addition to the usual superoxide dismutation activity, Cu,Zn-SOD has a peroxidative function that utilizes its own dismutation product, H202, as a substrate (17)(18)(19). During this reaction, the enzyme becomes inactivated in a relatively short period of time. However, this inactivation can be prevented by various anionic radical scavengers (18)(19)(20). Previous investigations from our laboratory (18,19) have shown that, during these reactions, free radicals, such as the hydroxyl radical ('OH), and scavenger-derived radicals are generated and some of these free radicals can escape from the active channel to the bulk solution. Because the enzymic activity is protected by small anions against H202 inactivation and concomitant generation of the anion-derived radicals, Cu,Zn-SOD behaves as an enzyme that catalyzes the formation of free radicals using anionic scavengers and H202 as substrates (18,19). These anionic scavengers, which become free radicals in this peroxidative reaction, include some neurotransmitters such as glutamate and taurine (19).We investigated whether there are any differences in the free radical-generating function between the wild-type and FALS mutant Cu,Zn-SOD. For this purpose, we cloned the wild-type and G93A cDNA of human Cu,Zn-SOD, overexpressed them in insect cells (Sf9), purified proteins, and measured their enzymic activities. Our results revealod-thatthe G93A and the wild-type Cu,Zn-SOD have identical dismutation activities. However, the free radical-generating function of the G93A mutant measured by the spin trapping method is enhanced relative to that of the wild-type enzyme. We found that this enhancement is due to a small decrease in the value of Km for H202. This gain-of-function, in part, may provide an explanation for the association of the FALS Cu,Zn-SOD mutants to ALS (21).

Exacerbation of Copper Toxicity in Primary Neuronal Cultures Depleted of Cellular Glutathione

“…In some familial ALS cases, mutations to the C u binding site of CdZn superoxide dismutase (SOD; superoxide oxidoreductase, EC 1.15.1 . l ) may increase the level of Cu-associated toxicity in motor neurons (Wiedau-Pazos et al, 1996;Yim et al, 1996;Ghadge et al, 1997;Zu et al, 1997).…”

Pro-oxidant activity of Cu,Zn-superoxide dismutase

“…In the presence of H202 and radical scavengers, especially with anionic character, both free 9 radicals and scavenger-derived radicals are produced (15,16). We also found that the free radical-generating function of several FALS-associated Cu,Zn-SOD mutants is enhanced relative to that of the wild-type enzyme, while the dismutation activities remain unchanged (17,18).…”

“…In the presence of H202 and radical scavengers, especially with anionic character, both free 9 radicals and scavenger-derived radicals are produced (15, 16). We also found that the free radical-generating function of several FALS-associated Cu,Zn-SOD mutants is enhanced relative to that of the wild-type enzyme, while the dismutation activities remain unchanged (17,18).To monitor the production of free radicals, two spin traps, 5,5-dimethyl-l-pyrroline N-oxide (DMPO) and Ntert-butyl-c~-phenylnitrone (PBN), were used in our studies (15) to convert transient free radicals to stable free radicals adducts according to the following reactions:The nature of the trapped free radical was identified by EPR spectroscopy. When O2-" (dissolved KO 2 in dried dimethylsulfoxide) was added to the NaHCOJCO 2 buffer at pH 7.6 containing 100 mM DMPO, EPR OH adducts.…”

“…Therefore, the enhanced free radical-generating activity induced by a burst of H202 formation, elevated level of Cu,Zn-SOD, or mutation of the enzyme may exert the deleterious effects in vivo. We demonstrated such an effect in FALS (17,18). Most of the FALS mutants have point-mutation sites in conserved interaction regions critical to the subunit fold and dimer contact, rather than residues in the active-site or in the electrostatic active channel (19)(20).…”

The role of metabolism in aging

“…The oxidative damage to proteins, lipids and DNA are all detectable and have been shown to vary among individuals, species and phylogenetic groups and to increase with age. Damage inflicted by ROS is believed to be implicated as the cause of a number of degenerative diseases including Parkinson's disease, amyotrophic lateral sclerosis (Lou Gehrig's disease) and others (Yim et al, 1996;Wallace, 1996;Simonian and Coyle, 1996). Though the activity in antioxidant enzymes may not correspond to its predicted role in aging (Matsuo et al, 1992), the "free radical" hypothesis clearly has a reasonable basis in documentation of the disastrous effects of oxygen use .…”