Figure 2:

Burst kinetics of the MDCC-labeled mutant T7 DNA polymerase. The time dependence of nucleotide incorporation was examined using quench-flow methods to evaluate the effects of the mutations and MDCC-labeling on the polymerase activity. (A) Amount of active enzyme after MDCC labeling was determined by an active-site titration experiment. The enzyme (200 nM, determined by a Bio-Rad protein assay with BSA standards) was preincubated with different amounts of DNA substrate. The reactions were then started by mixing the complex with 200 μM dCTP and were quenched after 18 ms. The amount of product formed as a function of the DNA concentration was fitted to a quadratic equation, ((E₀ + K_d + DNA) − ((E₀ + K_d + DNA)² − (4E₀DNA))^0.5)/2, to obtain the active enzyme concentration, E₀ = 92 ± 2.0 nM (92% active) and the dissociation constant for DNA binding, K_d = 36 ± 5.7 nM. (B) Nucleotide concentration dependence of the rate of polymerization was obtained by mixing a preformed enzyme–DNA complex (100 nM enzyme and 300 nM DNA after mixing) with various concentrations of dCTP. At each concentration, the time dependence of product formation was fit to a burst equation ([product] = A(1 – exp(−k₁t)) + k₂t) by nonlinear regression to derive the rate and amplitude of product formation. The concentration dependence of the rate of product formation (shown) was fit to a hyperbolic equation ([rate] = (k_polK_d)/(K_d + S)) to yield the smooth line defining a maximal burst rate of k_pol = 234 ± 9.4 s⁻¹ for dCTP incorporation and an apparent K_d = 24 ± 3.1 μM.