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⇌_kon ^koff2M_holo⇌^km-_km+2M _apo→^kaggA 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, Kd ≈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, k_m^- ≈ 1 × 10^-3 s^-1, k_m⁺ ≈ 1 × 10⁴ M ^-1·^s-1; 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 × 10 ^-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.