Evidence for Multiple Imino Intermediates and Identification of Reactive Nucleophiles in Peptide-Catalyzed -Eliminati
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- 作者：Andrew J. Kurtz ; M. L. Dodson ; and R. Stephen Lloyd
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：June 4, 2002
- 年：2002
- 卷：41
- 期：22
- 页码：7054 - 7064
- 全文大小：384K
- 年卷期：v.41,no.22(June 4, 2002)
- ISSN：1520-4995
文摘
Prior investigations have demonstrated that peptides containing a single aromatic residue flankedby basic ones, such as Lys-Trp-Lys, can incise the phosphodiester backbone of duplex DNA at an AP sitevia 
mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle">-elimination. An amine serves as the reactive nucleophile to attack C1' on the ring-open deoxyribosesugar to form a transient peptide-DNA imino (Schiff base) intermediate, which may be isolated as astable covalent species under reducing conditions. In the current study, we use this methodology todemonstrate that peptide-catalyzed mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle">-elimination proceeds via the formation of two Schiff baseintermediates, one of which was covalently trapped prior to strand incision and the other following strandincision. N-Terminal acetylation of reactive peptides significantly inhibited formation of a trapped Schiffbase complex; thus, we demonstrate for the first time that the preferred reactive nucleophile for peptidescatalyzing strand incision is the N-terminal mages/gifchars/alpha.gif" BORDER=0>-amino group, not an mages/gifchars/epsilon.gif" BORDER=0 >-amino group located on a lysineresidue as previously postulated. Trapping reactions in which the central tryptophan residue was changedto alanine did not have a significant impact on the efficiency of Schiff base formation, indicating that thepresence of an aromatic residue is dispensable for the step prior to peptide-catalyzed mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle">-elimination.Interestingly, the methodology presented here affords a convenient means for covalently attaching anarray of peptides onto AP site-containing DNA in a site-specific fashion. We suggest that the generationof such DNA-peptide cross-links may provide utility in studying the repair of biologically significantDNA-protein cross-link damage as DNA-peptide complexes may mimic intermediate structures alonga repair pathway for DNA-protein cross-links.