The rate and equilibrium constants for a multistep reaction sequence for the aggregation of superoxide dismutase in amyotrophic lateral sclerosis

Mutation-induced aggregation of the dimeric enzyme Cu, Zn superoxide dismutase 1 (SOD1) has been implicated in the familial form of the disease amyotrophic lateral sclerosis, but the mechanism of aggregation is not known. Here, we show that in vitro SOD1 aggregation is a multistep reaction that minimally consists of dimer dissociation, metal loss from the monomers, and oligomerization of the apo-monomers: D-holo reversible arrow(Kon)(Koff) 2M(holo) reversible arrow(Km+)(Km-) 2M(apo)-->(Kagg) A, where D-holo, M-holo, M-apo, and A are the holo-dimer, holo-monomer, apo-monomer, and aggregate, respectively. Under aggregation-promoting conditions (pH 3.5), the rate and equilibrium constants corresponding to each step are: (i) dimer dissociation, K-d approximate to1 muM; k(off) 1 x 10(-3) s(-1), k(on) approximate to1 x 10(3) M(-1.)s(-1); (ii) metal loss, K-m approximate to0.1 muM, k(m)(-) approximate to1 x 10(-3) s(-1), k(m)(+) approximate to1 x 10(4) M(-1.)s(-1); and (iii) assembly M (rate-limiting step), k(agg) approximate to1 x 10(3) M(-1.)s(-1). In contrast, under near-physiological conditions (pH 7.8), where aggregation is drastically reduced, dimer dissociation is less thermodynamically favorable: K-d approximate to0.1 nM, and extremely slow: k(off) approximate to3 x 10(-5) s(-1), k(on) approximate to3 x 10(5) M(-1.)s(-1). Our results suggest that familial amyotrophic lateral sclerosis-linked SOD1 aggregation occurs by a mutation-induced increase in dimer dissociation and/or increase in apo-monomer formation.