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. 2014 Dec 5;53(51):8061–8076. doi: 10.1021/bi501216a

Table 1. Complementary dNTP or rNTP Binding Rates for Wild-Type Φ29 DNAP and Mutantsa.

enzyme DNA ligand kon (s–1 μM–1)b koff (s–1)c koff (s–1)/kon (s–1 μM–1) Kd (μM)d
wt DNA1-H_H dGTP 21.4 ± 0.6 31.9 ± 0.5 1.49 ± 0.03 1.31 ± 0.04
Y254V DNA1-H_H dGTP 19.7 ± 0.9 140 ± 4 7.3 ± 0.3 6.59 ± 0.2
D12A/D66A DNA1-H_H dGTP 16.7 ± 0.4 17.7 ± 0.3 1.02 ± 0.02 0.99 ± 0.04
D12A/D66A/Y254V DNA1-H_H dGTP 32.4 ± 1.0 126 ± 2.3 3.9 ± 0.2 4.76 ± 0.17
wt DNA1-H_H rGTP >3.2 >5400 e 1702 ± 239
Y254V DNA1-H_H rGTP 8.9 ± 1.8 777 ± 16 91 ± 24 83.6 ± 3.6
D12A/D66A DNA1-H_H rGTP >7.1 >4900 e 695 ± 32
D12A/D66A/Y254V DNA1-H_H rGTP 12.6 ± 1.9 546 ± 32 45.2 ± 4.2 53.1 ± 1.6
wt DNA1-OH_H dGTP 8.9 ± 1.8 526 ± 28 60.8 ± 9.1 59.4 ± 7.3
Y254V DNA1-OH_H dGTP 12.7 ± 0.3 1126 ± 39 85.7 ± 1.8 86.4 ± 6.1
D12A/D66A DNA1-OH_H dGTP 9.9 ± 0.6 318 ± 26 32 ± 1.0 27.2 ± 1.7
D12A/D66A/Y254V DNA1-OH_H dGTP 14.8 ± 0.2 897 ± 19 58.8 ± 1.3 63.4 ± 2.6
a

Rates were determined using dwell time samples extracted from ionic current traces (see Methods section) and a three-state kinetic model (consisting of transitions r1, r2, kon and koff in the model diagram in Figure 1a). Experiments were conducted at 180 mV; kon[dNTP] and koff are independent of the applied voltage.38 All values are reported with the standard error.

b

The dNTP or rNTP association rate constant.

c

The dNTP or rNTP dissociation rate.

d

Kd values are determined from the vertical intercepts of the fitting lines to the log–log plot of normalized p/(1 – p) – 1 vs [dGTP] or [rGTP], where p is equilibrium probability of the lower amplitude level (see the model diagram in Figure 1a). The plots of normalized p/(1 – p) – 1 vs [dGTP] or [rGTP] are shown in Figures 5 and 7.

e

Not determined.