| Abstract
| - The core of DNA polymerase III, the replicative polymerase in Escherichia coli, consists ofthree subunits (α, ε, and θ). The ε subunit is the 3‘−5‘ proofreading exonuclease that associates with thepolymerase (α) through its C-terminal region and θ through a 185-residue N-terminal domain (ε186). Aspectrophotometric assay for measurement of ε activity is described. Proteins ε and ε186 and the ε186·θcomplex catalyzed the hydrolysis of the 5‘-p-nitrophenyl ester of TMP (pNP-TMP) with similar values ofkcat and KM, confirming that the N-terminal domain of ε bears the exonuclease active site, and showingthat association with θ has little direct effect on the chemistry occurring at the active site of ε. On theother hand, formation of the complex with θ stabilized ε186 by ∼14 °C against thermal inactivation. Forε186, kcat = 293 min-1 and KM = 1.08 mM at pH 8.00 and 25 °C, with a Mn2+ concentration of 1 mM.Hydrolysis of pNP-TMP by ε186 depended absolutely on divalent metal ions, and was inhibited by theproduct TMP. Dependencies on Mn2+ and Mg2+ concentrations were examined, giving a KMn of 0.31mM and a kcat of 334 min-1 for Mn2+ and a KMg of 6.9 mM and a kcat of 19.9 min-1 for Mg2+. Inhibitionby TMP was formally competitive [Ki = 4.3 μM (with a Mn2+ concentration of 1 mM)]. The pH dependenceof pNP-TMP hydrolysis by ε186, in the pH range of 6.5−9.0, was found to be simple. KM was essentiallyinvariant between pH 6.5 and 8.5, while kcat depended on titration of a single group with a pKa of 7.7,approaching limiting values of 50 min-1 at pH <6.5 and 400 min-1 at pH >9.0. These data are used inconjunction with crystal structures of the complex of ε186 with TMP and two Mn(II) ions bound at theactive site to develop insights into the mechanisms of pNP-TMP hydrolysis by ε at high and low pHvalues.
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