Hydrolysis of the 5′-p-nitrophenyl ester of TMP by the proofreading exonuclease (ε) subunit of Escherichia coli DNA polymerase III

The core of DNA polymerase III, the replicative polymerase in Escherichia coli, consists of three subunits (alpha, epsilon, and theta). The 6 subunit is the 3'-5' proofreading exonuclease that associates with the polymerase (alpha) through its C-terminal region and theta through a 185-residue N-terminal domain (6186). A spectrophotometric assay for measurement of epsilon activity is described. Proteins epsilon and epsilon186 and the epsilon186.theta complex catalyzed the hydrolysis of the 5'-p-nitrophenyl ester of TMP (pNP-TMP) with similar values of k(cat) and K-M, confirming that the N-terminal domain of epsilon bears the exonuclease active site, and showing that association with theta has little direct effect on the chemistry occurring at the active site of epsilon. On the other hand, formation of the complex with theta stabilized epsilon186 by similar to14 degreesC against thermal inactivation. For epsilon186, k(cat) = 2193 min(-1) and K-M = 1.08 mM at pH 8.00 and 25 degreesC, with a Mn2+ concentration of 1 mM. Hydrolysis of pNP-TMP by 6 186 depended absolutely on divalent metal ions, and was inhibited by the product TMP. Dependencies on Mn2+ and Mg2+ concentrations were examined, giving a K-Mn of 0.31 mM and a k(cat) of 334 min(-1) for Mn2+ and a K-Mg of 6.9 mM and a k(cat) of 19.9 min(-1) for Mg2+. Inhibition by TMP was formally competitive [K-i = 4.3 muM (with a Mn2+ concentration of 1 mM)]. The pH dependence of pNP-TMP hydrolysis by epsilon186, in the pH range of 6.5-9.0, was found to be simple. Km was essentially invariant between pH 6.5 and 8.5, while k(cat) depended on titration of a single group with a pK(a) 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 in conjunction with crystal structures of the complex of E 186 with TMP and two Mn(II) ions bound at the active site to develop insights into the mechanisms of pNP-TMP hydrolysis by 6 at high and low pH values.