The rate and equilibrium constants for a multistep reaction sequence for the aggregation of superoxide dismutase in amyotrophic lateral sclerosis - PubMed (original) (raw)
The rate and equilibrium constants for a multistep reaction sequence for the aggregation of superoxide dismutase in amyotrophic lateral sclerosis
Sagar D Khare et al. Proc Natl Acad Sci U S A. 2004.
Abstract
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.
Figures
Fig. 1.
Size-exclusion profiles of SOD1 at pH 7.8. (A) The calibration curve for the column obtained by using globular protein standards at pH 7.8 was log (_M_m/1,000) = 4.56–1.526·(_V_e/_V_0), where _V_e is the elution volume and _V_0 is the void volume (7.8 ml) of the column. SOD dimer (32 kDa) and monomer (16 kDa) were expected to elute at 15.5 and 17.0 ml, respectively. (B) Elution profile at 30 μM: SOD1 elutes at 15.6 ml and thus is dimeric. (C) Immunostaining dot-blot profile of a 5 nM SOD1 sample after 24 h of incubation. The peaks are Gaussian fits and correspond to the dimer and monomer.
Fig. 2.
The dimer dissociation rates of SOD1 measured by SPR. (A) Scheme for SPR measurement of SOD1 dimer dissociation rate. The rate constant at pH 3.5 was calculated as 1.07 × 10–3 s–1 (B) and 3.13 × 10–5 s–1 at pH 7.8 (C), by fitting to a single exponential (dotted lines). The initial rate was followed in the pH range from 2.5 to 12.7. (D) For dissociation at pH 6.6, the rate constant was found to be 2.75 × 10–5 for bovine SOD1 and 1.53 × 10–5 for human SOD1 (Inset). (E) The _k_off dependence on pH follows a titration curve with apparent pKa values of <2.5 and >12.5 at the acidic and basic ends of the pH range respectively. The lines have slopes of 1 and –1 (Eq. 2).
Fig. 3.
Kinetics of aggregation of SOD1. (A) Aggregation at 30 μM. SOD1 elution profiles were obtained at initial time and at 24 and 48 h by immunostaining dot blots and UV absorbance (solid lines). The profiles were normalized according to the fraction with the highest signal intensity, and the monomer is more pronounced in the dot-blot elution profile. Aggregate is defined as the protein not eluting as dimer or monomer. (B) Aggregation at 1 μM. SOD1 was detected by immunobloting column fractions with Gaussian fits to the spot-intensity profile. (C) Dot blots showing the elution profile at 30 and 1 μM before and after incubation. The fractions corresponding to the dimer and monomer at 30 μM were diluted 30-fold to ensure linearity of signal. (D) Time course of 30 μM SOD1 under dialysis conditions. (Inset) An image of the fibrilar product after 21 h, obtained by negative staining electron microscopy. (E) Fit of the model in Eq. 3 to the results obtained in D. The continuous curves are obtained from numerical simulations with the optimized rate constants. (F) Predicted aggregation profiles with 30 and 1 μM using the parameters derived in E. The predicted fraction of aggregates in 30 and 1 μM SOD1 after 48 h was 14% and 63%, respectively, in close agreement with the experimentally measured values in A and B.
Fig. 4.
A scheme for the aggregation of SOD1. The more rapid aggregation at pH 3.5 compared with pH 7.8 results from the increased _K_d and _k_off values at pH 3.5. Exposure to low pH decreases the stability of SOD1 by 5.2 (=13.5–8.3) kcal/mol, whereas the dissociation barrier is reduced by ≈2 kcal/mol. At pH 3.5, the dissociation free energy of Zn from SOD1 monomer, _M_holo, is ≈9.6 kcal/mol. In the minimal reaction sequence (Eq. 1), the apo-monomers, _M_apo, aggregate to form insoluble fibrils; this step is the rate-limiting step for aggregation.
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