Table I.
Kinetic and hydrodynamic a parameters of Dnm1 and Dnm1 mutants
| Parameters | Wild-type Dnm1 (high ionic strength)b |
Wild-type Dnm1 (low ionic strength)b |
Dnm1 1–388 (GTPase domain) |
Dnm1G385D |
|---|---|---|---|---|
| kcat (min−1)c | 11.6 | 50.7 | 0.84 | 3.11 |
| K0.5/Km (μM)c | 214 | 79.1 | 19.2 | 109.9 |
| Hill coefficient | 3.3 | 1.6 | 1 | 1 |
| Kinetic lag | + | + | − | − |
| Ve (mL)d | 9.54 | ND | 16.9 | 13.1 |
| Stokes' radius (A)e | 130 | ND | 33.0 | 73.2 |
| Sedimentation coefficient (S)f |
18.6 | ND | 2.7 | 5.7 |
| Molecular mass (kD)g |
995 | ND | 36.6 | 174 |
| Estimated number of subunitsh , i |
10 | ND | 1 | 2 |
a
Dnm1G385D and GTPase domain at 500 mM NaCl. Wild-type Dnm1 at 750 mM NaCl.
b
High ionic strength, 500 mM NaCl; low ionic strength, 150 mM NaCl.
c
Calculated by nonlinear least squares method using the GenFit function of Mathcad.
d
Superose 6 column.
e
Calculated based on Porath correlation of gel filtration standards.
f
Calculated based on native high molecular mass markers.
g
Estimated by using the method developed by Siegel and Monty (1966).
h
Subunit estimation based on the unmodified molecular mass of the protein, which was determined by mass spectrometry (87 kD).
i
Rounded to nearest subunit. Actual values are as follows: Dnm1G385D, 2.0; Dnm1 GTPase domain, 0.92; and wild-type Dnm1, 10.4.