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Reaction intermediates in the catalytic mechanism of escherichia coli muty DNA glycosylase

Academic Article
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Overview

authors

  • Manuel, R. C.
  • Hitomi, K.
  • Arvai, A. S.
  • House, P. G.
  • Kurtz, A. J.
  • Dodson, M. L.
  • McCullough, A. K.
  • Tainer, John
  • Lloyd, R. S.

publication date

  • November 2004

journal

  • Journal of Biological Chemistry  Journal

abstract

  • The Escherichia coli adenine DNA glycosylase, MutY, plays an important role in the maintenance of genomic stability by catalyzing the removal of adenine opposite 8-oxo-7,8-dihydroguanine or guanine in duplex DNA. Although the x-ray crystal structure of the catalytic domain of MutY revealed a mechanism for catalysis of the glycosyl bond, it appeared that several opportunistically positioned lysine side chains could participate in a secondary beta-elimination reaction. In this investigation, it is established via site-directed mutagenesis and the determination of a 1.35-A structure of MutY in complex with adenine that the abasic site (apurinic/apyrimidinic) lyase activity is alternatively regulated by two lysines, Lys142 and Lys20. Analyses of the crystallographic structure also suggest a role for Glu161 in the apurinic/apyrimidinic lyase chemistry. The beta-elimination reaction is structurally and chemically uncoupled from the initial glycosyl bond scission, indicating that this reaction occurs as a consequence of active site plasticity and slow dissociation of the product complex. MutY with either the K142A or K20A mutation still catalyzes beta and beta-delta elimination reactions, and both mutants can be trapped as covalent enzyme-DNA intermediates by chemical reduction. The trapping was observed to occur both pre- and post-phosphodiester bond scission, establishing that both of these intermediates have significant half-lives. Thus, the final spectrum of DNA products generated reflects the outcome of a delicate balance of closely related equilibrium constants.

subject areas

  • Adenine
  • Aspartic Acid
  • Binding Sites
  • Catalysis
  • Catalytic Domain
  • Crystallography, X-Ray
  • DNA
  • DNA Glycosylases
  • Dose-Response Relationship, Drug
  • Escherichia coli
  • Glutamic Acid
  • Guanine
  • Kinetics
  • Lysine
  • Models, Chemical
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Mutation
  • Protein Conformation
  • Time Factors
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Identity

International Standard Serial Number (ISSN)

  • 0021-9258

Digital Object Identifier (DOI)

  • 10.1074/jbc.M403944200

PubMed ID

  • 15326180
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Additional Document Info

start page

  • 46930

end page

  • 46939

volume

  • 279

issue

  • 45

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