Hydrolysis of the 5'-p-nitrophenyl ester of TMP by the proofreading exonuclease (E) subunit of Escherichia coli DNA polymerase IIIFaculty of Science, Medicine and Health - Papers
AbstractThe core of DNA polymerase III, the replicative polymerase in Escherichia coli, consists of three subunits (α, ε, and θ). The ε subunit is the 3‘−5‘ proofreading exonuclease that associates with the polymerase (α) through its C-terminal region and θ through a 185-residue N-terminal domain (ε186). A spectrophotometric assay for measurement of ε activity is described. Proteins ε and ε186 and the ε186·θ complex catalyzed the hydrolysis of the 5‘-p-nitrophenyl ester of TMP (pNP-TMP) with similar values of kcat and KM, confirming that the N-terminal domain of ε bears the exonuclease active site, and showing that association with θ has little direct effect on the chemistry occurring at the active site of ε. On the other hand, formation of the complex with θ stabilized ε186 by 14 °C against thermal inactivation. For ε186, 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 the product TMP. Dependencies on Mn2+ and Mg2+ concentrations were examined, giving a KMn of 0.31 mM 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+. Inhibition by TMP was formally competitive [Ki = 4.3 μM (with a Mn2+ concentration of 1 mM)]. The pH dependence of pNP-TMP hydrolysis by ε186, 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 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 in conjunction with crystal structures of the complex of ε186 with TMP and two Mn(II) ions bound at the active site to develop insights into the mechanisms of pNP-TMP hydrolysis by ε at high and low pH values.
Citation InformationSamir Hamdan, Esther M Bulloch, Philip R Thompson, Jennifer L Beck, et al.. "Hydrolysis of the 5'-p-nitrophenyl ester of TMP by the proofreading exonuclease (E) subunit of Escherichia coli DNA polymerase III" (2002)
Available at: http://works.bepress.com/nick_dixon/49/