Elementary steps in the DNA polymerase I reaction pathway

Floyd Ransom Bryant, K. A. Johnson, S. J. Benkovic

Research output: Contribution to journalArticle

Abstract

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 fcon = 1.2 × 106 M-1 s-1, koff = 0-25 s-1, and KD = 2 × 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°C) and dissociation of the enzyme from the template-primer under steady-state conditions (koff ss = 0.1 s-1). The rapid quench time course of the polymerization reaction (kcat = 2.5 s-1, 20 °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.

Original languageEnglish (US)
Pages (from-to)3537-3546
Number of pages10
JournalBiochemistry®
Volume22
Issue number15
StatePublished - 1983
Externally publishedYes

Fingerprint

DNA Polymerase I
Polymerization
Isotopes
Chemical bonds
Isomerization
Homopolymerization
Catalysis
Mental Competency
Escherichia coli
Polymers
Experiments
Molecules
Substrates
Enzymes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Bryant, F. R., Johnson, K. A., & Benkovic, S. J. (1983). Elementary steps in the DNA polymerase I reaction pathway. Biochemistry®, 22(15), 3537-3546.

Elementary steps in the DNA polymerase I reaction pathway. / Bryant, Floyd Ransom; Johnson, K. A.; Benkovic, S. J.

In: Biochemistry®, Vol. 22, No. 15, 1983, p. 3537-3546.

Research output: Contribution to journalArticle

Bryant, FR, Johnson, KA & Benkovic, SJ 1983, 'Elementary steps in the DNA polymerase I reaction pathway', Biochemistry®, vol. 22, no. 15, pp. 3537-3546.
Bryant, Floyd Ransom ; Johnson, K. A. ; Benkovic, S. J. / Elementary steps in the DNA polymerase I reaction pathway. In: Biochemistry®. 1983 ; Vol. 22, No. 15. pp. 3537-3546.
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