HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 282, Issue 42, 30577-30585, October 19, 2007

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 282/42/30577    most recent M704341200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Maher, R. L. Articles by Bloom, L. B. Search for Related Content PubMed PubMed Citation Articles by Maher, R. L. Articles by Bloom, L. B. Social Bookmarking                      What's this?

Pre-steady-state Kinetic Characterization of the AP Endonuclease Activity of Human AP Endonuclease 1^*

From the Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610-0245

Human AP endonuclease 1 (APE1, REF1) functions within the base excision repair pathway by catalyzing the hydrolysis of the phosphodiester bond 5 ' to a baseless sugar (apurinic or apyrimidinic site). The AP endonuclease activity of this enzyme and two active site mutants were characterized using equilibrium binding and pre-steady-state kinetic techniques. Wild-type APE1 is a remarkably potent endonuclease and highly efficient enzyme. Incision 5 ' to AP sites is so fast that a maximal single-turnover rate could not be measured using rapid mixing/quench techniques and is at least 850 s^–1. The entire catalytic cycle is limited by a slow step that follows chemistry and generates a steady-state incision rate of about 2 s^–1. Site-directed mutation of His-309 to Asn and Asp-210 to Ala reduced the single turnover rate of incision 5 ' to AP sites by at least 5 orders of magnitude such that chemistry (or a step following DNA binding and preceding chemistry) and not a step following chemistry became rate-limiting. Our results suggest that the efficiency with which APE1 can process an AP site in vivo is limited by the rate at which it diffuses to the site and that a slow step after chemistry may prevent APE1 from leaving the site of damage before the next enzyme arrives to continue the repair process.

Received for publication, May 25, 2007 , and in revised form, August 24, 2007.

^* This work was supported by National Institutes of Health GM055596 (to L. B. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains a supplemental figure.

¹ To whom correspondence should be addressed. Tel.: 352-392-8708; Fax: 352-392-6511; E-mail: lbloom{at}ufl.edu.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				S. Adhikari, A. Uren, and R. Roy Dipole-Dipole Interaction Stabilizes the Transition State of Apurinic/Apyrimidinic Endonuclease Abasic Site Interaction J. Biol. Chem., January 18, 2008; 283(3): 1334 - 1339. [Abstract] [Full Text] [PDF]