Hydrolysis of the 5'-p-Nitrophenyl Ester of TMP by the Proofreading Exonuclease () Subunit of Escherichia coli DNA P

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• 作者：Samir Hamdan ; Esther M. Bulloch ; Phillip R. Thompson ; Jennifer L. Beck ; Ji Yeon Yang ; Jeffrey A. Crowther ; Penelope E. Lilley ; Paul D. Carr ; David L. Ollis ; Susan E. Brown ; and Nicholas E. Dixon
• 刊名：Biochemistry
• 出版年：2002
• 出版时间：April 23, 2002
• 年：2002
• 卷：41
• 期：16
• 页码：5266 - 5275
• 全文大小：118K
• 年卷期：v.41,no.16(April 23, 2002)
• ISSN：1520-4995

文摘

The core of DNA polymerase III, the replicative polymerase in Escherichia coli, consists ofthree subunits (s/gifchars/alpha.gif" BORDER=0>, s/gifchars/epsilon.gif" BORDER=0 >, and s/gifchars/theta.gif" BORDER=0 >). The s/gifchars/epsilon.gif" BORDER=0 > subunit is the 3'-5' proofreading exonuclease that associates with thepolymerase (s/gifchars/alpha.gif" BORDER=0>) through its C-terminal region and s/gifchars/theta.gif" BORDER=0 > through a 185-residue N-terminal domain (s/gifchars/epsilon.gif" BORDER=0 >186). Aspectrophotometric assay for measurement of s/gifchars/epsilon.gif" BORDER=0 > activity is described. Proteins s/gifchars/epsilon.gif" BORDER=0 > and s/gifchars/epsilon.gif" BORDER=0 >186 and the s/gifchars/epsilon.gif" BORDER=0 >186·s/gifchars/theta.gif" BORDER=0 >complex catalyzed the hydrolysis of the 5'-p-nitrophenyl ester of TMP (pNP-TMP) with similar values ofk_cat and K_M, confirming that the N-terminal domain of s/gifchars/epsilon.gif" BORDER=0 > bears the exonuclease active site, and showingthat association with s/gifchars/theta.gif" BORDER=0 > has little direct effect on the chemistry occurring at the active site of s/gifchars/epsilon.gif" BORDER=0 >. On theother hand, formation of the complex with s/gifchars/theta.gif" BORDER=0 > stabilized s/gifchars/epsilon.gif" BORDER=0 >186 by ~14 s/entities/deg.gif">C against thermal inactivation. Fors/gifchars/epsilon.gif" BORDER=0 >186, k_cat = 293 min^-1 and K_M = 1.08 mM at pH 8.00 and 25 s/entities/deg.gif">C, with a Mn²⁺ concentration of 1 mM.Hydrolysis of pNP-TMP by s/gifchars/epsilon.gif" BORDER=0 >186 depended absolutely on divalent metal ions, and was inhibited by theproduct TMP. Dependencies on Mn²⁺ and Mg²⁺ concentrations were examined, giving a K_Mn of 0.31mM 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⁺. Inhibitionby TMP was formally competitive [K_i = 4.3 s/entities/mgr.gif">M (with a Mn²⁺ concentration of 1 mM)]. The pH dependenceof pNP-TMP hydrolysis by s/gifchars/epsilon.gif" BORDER=0 >186, in the pH range of 6.5-9.0, was found to be simple. K_M was essentiallyinvariant 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 inconjunction with crystal structures of the complex of s/gifchars/epsilon.gif" BORDER=0 >186 with TMP and two Mn(II) ions bound at theactive site to develop insights into the mechanisms of pNP-TMP hydrolysis by s/gifchars/epsilon.gif" BORDER=0 > at high and low pHvalues.