TABLE 2.

Kinetic parameters of wild-type and mutant Pol I(KF) from stopped-flow fluorescence data with a 2-AP(+1) reporter

Data are the average values from two experiments and are reported as mean ± S.D.

Protein	Rate1a	Rate2	Amp1/Amp2b	Kdoverallc
	s−1	s−1		μm
WT	⩾400	65 ± 13	75:25	2.8 ± 0.6
D705A	⩾900	13 ± 4	90:10	8.6 ± 3.1
D882A	⩾400	22 ± 4	55:45	8.1 ± 1.2

^a Rate₁, Rate₂, Amp₁, and Amp₂ were calculated taking account of the amplitude that is lost in the instrument dead time (supplemental Fig. S3 and Table S2). Rate₂ showed very little variation with dNTP concentration, and therefore we report the average of Rate₂ values at all nucleotide concentrations.

^b The ratio of the maximum values of the amplitudes of the fast and slow phases, each determined by plotting amplitude against dGTP concentration and fitting the data to a hyperbolic equation.

^c K_{d overall} was determined by plotting the end point of each fluorescence trace against dGTP concentration and fitting to a hyperbolic equation (supplemental Fig. S3).