The steady state kinetic parameters and non-processivity of Escherichia coli deoxyribonucleic acid polymerase I.

Abstract:

A steady state kinetic study of Escherichia coli DNA polymerase I has been carried out using poly[d(A-T)] as the template-primer substrate. The results of substrate saturation and product inhibition kinetic studies suggest an altered Ordered Bi Bi mechanism for the enzyme. The Michaelis constants for polymer, d-atp, and dTTP are 5 nM (3'-OH ends), 1 muM, and 2 muM, respectively. The apparent equilibrium constant for the reaction, Keq equals [PPi]/[dNTP], was estimated as greater than or equal to 500. No quaternary complex of enzyme, template, and both deoxynucleoside triphosphates was detected. Single turnover experiments at 4 degrees indicated that the enzyme functions non-processively under the specified conditions, that is, dissociates after each catalytic step. The results at higher temperature were consistent with dissociation within 30 steps. Furthermore, at 4 degrees a burst of incorporation stoichiometric with the amount of enzyme was observed upon initiation of the reaction, indicating that the rate-limiting step in the steady state occurs after phosphodiester bond formation. There is a linear Arrhenius dependence of the initial reaction on temperature in the range 4-40 degrees, with an apparent Ea equals 17 kcal/mol. The rate equations appropriate for template-dependent polymerases which dissociate after each catalytic step have been derived.

Polymerases:

Topics:

Exonuclease Activity, Source / Purification, Historical Protein Properties (MW, pI, ...), Kinetic Parameters

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Eco Pol I McClure WR1975 3-5' Exonuclease (proofreading) Yes
Eco Pol I McClure WR1975 5-3' Exonuclease Yes
Eco Pol I McClure WR1975 Full length or truncated Full length
Eco Pol I McClure WR1975 Specific Activity 1.5E+04 units/mg
Eco Pol I McClure WR1975 KM 1uM Reaction: Nucleotide incorporation; Substrate: dATP
Eco Pol I McClure WR1975 KM 2uM Reaction: Nucleotide incorporation; Substrate: dTTP

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