Elementary steps in the DNA polymerase I reaction pathway.

Bryant FR, Johnson KA, Benkovic SJ
Biochemistry (1983), Volume 22, Page 3537
PubMed entry


The polymerization reaction catalyzed by Escherichia coli DNA ...
The polymerization reaction catalyzed by Escherichia coli DNA polymerase I (Pol I) has been studied by using the homopolymer template-primer system poly(dA).oligo(dT). Isotope-partitioning experiments indicate that the reaction follows an ordered mechanism in which Pol I first combines with template-primer to form an E.poly complex followed by addition of MgTTP and catalysis. The parameters governing the binding of Pol I to the template-primer are kon = 1.2 X 10(6) M-1 s-1, koff = 0.25 s-1, and KD = 2 X 10(-7) M. Efforts to demonstrate the catalytic competence of the binary E.MgTTP complex were unsuccessful. Following initiation of the catalytic cycle, Pol I catalyzes the incorporation of an average of 40-50 TTP molecules into polymer before dissociating from the template-primer. The processive nature of the polymerization reaction as reflected by the isotope-trapping time dependence can be accounted for by a model in which processive synthesis is treated as a simple partitioning between continued polymerization (kcat = 3.8 s-1, 22 degrees C) and dissociation of the enzyme from the template-primer under steady-state conditions (koffss = 0.1 s-1). The rapid quench time course of the polymerization reaction (kcat = 2.5 s-1, 20 degrees C) exhibited a pre-steady-state burst consistent with two partially rate-determining steps, one of which precedes the actual chemical phosphodiester bond-forming step (k = 4.6 s-1) and the other which follows this step (k = 4.0 s-1). Binding of MgTTP to the E.poly complex was shown to be a rapid equilibrium step by steady-state isotope-partitioning experiments. This suggested that the first rate-determining step may be a first-order isomerization which follows the binding of substrates and precedes bond formation.




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