Table 2. Kinetic Rate and Binding Constants for WT versus I260Q Nucleotide Incorporation into 19/36AP(T)/15 and 19/36AP(C)/15 DNA Substrates at pH 8.5^a.

	T:A (matched)		T:G (mismatched)
	wild-type	I260Q	wild-type	I260Q
k2	116 ± 5 s−1	108 ± 7 s−1	255 ± 9 s−1	485 ± 25 s−1
Kd	30 ± 4 μM	6.6 ± 2.1 μM	433 ± 72 μM	232 ± 37 μM
kpol	42.9 ± 0.6 s−1	43.6 ± 1.4 s−1	5.7 ± 0.1 s−1	13.6 ± 0.2 s−1
Kd,app	6.8 ± 0.5 μM	7.1 ± 1.2 μM	489 ± 26 μM	49 ± 3 μM
kquench	45.7 ± 2.8 s−1	47.6 ± 2.5 s−1	4.8 ± 0.3 s−1	14.8 ± 0.5 s−1
kpol/Kd,appb	6.3	6.2	0.012	0.28
fidelityc			541	23

	C:G (matched)		C:A (mismatched)
	wild-type	I260Q	wild-type	I260Q
kpol	20.8 ± 0.5 s−1	18.3 ± 0.4 s−1	1.9 ± 0.1 s−1	9.4 ± 0.2 s−1
Kd,app	2.2 ± 0.3 μM	1.2 ± 0.1 μM	227 ± 22 μM	45 ± 3 μM
kpol/Kd,appb	9.7	15	0.0082	0.21
fidelityc			1180	74

^aThe k₂, K_d, k_pol and K_d,app values were obtained from hyperbolic fit of the dNTP concentration dependence of the observed rates of the fast and slow fluorescence phases (k_fast and k_slow) as described in . The rate of dNTP incorporation (k_quench) was obtained from single exponential fit of rapid chemical quench data at a saturating dNTP concentration. The k₂ value represents the rate constant of forward conformational closing, while K_d reflects the stability of the ternary complex before closing. Therefore, WT and I260Q show little difference during the initial dNTP binding before conformational change. The k_pol value represents the maximum rate of dNTP incorporation, while the K_d,app value possesses a contribution from all steps up to the rate-limiting step and can be thought as the dissociation constant of the closed ternary complex.

^bCatalytic efficiency as measured in units of μM⁻¹ s⁻¹.

^cFidelity defined as [(k_pol/K_d,app)_cor + (k_pol/K_d,app)_inc]/(k_pol/K_d,app)_inc, where the subscripts “cor” and “inc” indicate correct (matched) and incorrect (mismatched) nucleotide incorporation, respectively.