TABLE 3.
Kinetic parameters of wild-type and D705A Pol I(KF) in the stopped-flow FRET-based assay of the fingers-closing conformational change
| Protein | Rate1a | Rate2a | Amp1:Amp2b | kmaxc | Kd(app)c | Kdoveralld |
|---|---|---|---|---|---|---|
| s−1 | s−1 | s−1 | μm | μm | ||
| Mg2+ as metal cofactor | ||||||
| WT | 75 ± 18 | 6.3 ± 1.8 | 5.1 | 110 | 19 | 8.6 |
| D705A | ⩾500e | 110 ± 4 | ∼6 | 120 ± 40 | ||
| Mn2+ as metal cofactor | ||||||
| WT | 91 ± 6 | 6.3 ± 2.3 | 130 | 19 | <1 | |
| D705A | 50 ± 12 | 60 ± 6 | 18 ± 7 | 6.9 ± 0.2 | ||
a The individual rates reported for WT Pol I(KF) were measured at 50 μm dTTP, and those for the D705A mutant were measured at 1 mm dTTP (in Mg2+) and 100 μm dTTP (in Mn2+). Values reported as mean ± S.D were from at least two independent experiments; the others were from single determinations. Examples of curve fitting and residuals for single and double exponentials are shown in supplemental Fig. S5.
b The ratio of the maximum values of the amplitudes of the fast and slow phases, determined by plotting the individual amplitudes against dTTP concentration and fitting the data to a hyperbolic equation.
c The parameters kmax and Kd(app) were derived from rate measurements at a series of dTTP concentrations by plotting Rate1 against dTTP concentration and fitting the data to a hyperbolic equation.
d Kd overall was determined from the hyperbolic dependence on dTTP concentration of either the total amplitude or the end point of the fluorescence traces.
e The rate of this very fast process was estimated assuming that 80% of the process was accomplished within 3 ms.