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: 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 ≈1 μM; k_off ≈ 1 × 10^-3 s^-1, k_on ≈1 × 10³ M^-1.s^-1; (ii) metal loss, K_m ≈0.1 μM, , ; and (iii) assembly (rate-limiting step), k_agg ≈1 × 10³ 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 ≈ 0.1 nM, and extremely slow: k_off ≈3 × 10^-5 s^-1, k_on ≈3 × 10⁵ 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 apomonomer formation.