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: [reaction: see text], 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 approximately 1 microM; k(off) approximately 1 x 10(-3) s(-1), k(on) approximately 1 x 10(3) M(-1).s(-1); (ii) metal loss, Km approximately 0.1 microM, km- approximately 1 x 10(-3)s(-1), km+ approximately 1 x 10(4) M(-1).s(-1); and (iii) assembly (rate-limiting step), k(agg) approximately 1 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: Kd approximately 0.1 nM, and extremely slow: k(off) approximately 3 x 10(-5) s(-1), k(on) approximately 3 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 apomonomer formation.
Khare
Sagar D
SD
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599, USA.
Caplow
Michael
M
Dokholyan
Nikolay V
NV
D000690:Amyotrophic Lateral Sclerosis / Q000201:enzymology* / Q000235:genetics; D001051:Apoenzymes / Q000737:chemistry / Q000378:metabolism; D019281:Dimerization; D020035:Holoenzymes / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D046911:Macromolecular Substances; D008670:Metals / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D020836:Protein Structure, Quaternary; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000072105:Superoxide Dismutase-1; D013816:Thermodynamics
D001051:Apoenzymes; D020035:Holoenzymes; D046911:Macromolecular Substances; D008670:Metals; C000606290:SOD1 protein, human; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1