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J. Biol. Chem., Vol. 280, Issue 4, 2714-2720, January 28, 2005

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Kinetic Analysis of the Individual Steps of Protein Splicing for the Pyrococcus abyssi PolII Intein^*

Kenneth V. Mills, Deirdre M. Dorval, and Katherine T. Lewandowski

From the College of the Holy Cross, Department of Chemistry, Worcester, Massachusetts 01610

Protein splicing involves the excision of an intervening polypeptide, the intein, from flanking polypeptides, the exteins, concomitant with the specific ligation of the exteins. The intein that interrupts the DNA polymerase II DP2 subunit in Pyrococcus abyssi can be overexpressed and purified as an unspliced precursor, which allows for a detailed in vitro kinetic analysis of the individual steps of protein splicing. The first order rate constant for splicing of this intein, which has a non-canonical Gln at its C terminus, is 9.3 x 10^-6 s^-1 at 60 °C. The rate constant for splicing increases 3-fold with substitution of Asn for the C-terminal Gln. The pseudo first order rate constant of dithiothreitol-dependent N-terminal cleavage is 1 x 10^-4 s^-1. The first order rate constant of C-terminal cleavage is 1.2 x 10^-5 s^-1 with Gln at the C-terminal position, 2.8 x 10^-4 s^-1 with Asn, and decreases significantly with mutation of the penultimate His of the intein to Ala. N-terminal cleavage is most efficient between pH 7 and 7.5 and decreases at both more acidic and alkaline pH values, whereas C-terminal cleavage and splicing are both efficient over a broader range of pH values.

Received for publication, November 1, 2004 , and in revised form, November 22, 2004.

^* This work was supported by the Donors of the Petroleum Research Fund, administered by the American Chemical Society, the Research Corporation via a Cottrell College Science Award, and the National Science Foundation (DBI-0320824). 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.

Both authors contributed equally to this work.

To whom correspondence should be addressed: College of the Holy Cross, Dept. of Chemistry, 1 College St., Worcester, MA 01610. Tel.: 508-793-3380; Fax: 508-793-3530; E-mail: kmills{at}holycross.edu.

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