Evidence for an Imino Intermediate in the T4 Endonuclease V Reaction

M. L. Dodson, R. Stephen Lloyd, Robert D. Schrock

Research output: Contribution to journalArticle

133 Scopus citations

Abstract

Reductive methylation and site-directed mutagenesis experiments have implicated the N-terminal α-amino group of T4 endonuclease V in the glycosylase and abasic lyase activities of the enzyme. NMR studies have confirmed the involvement of the N-terminal α-amino group in the inhibition of enzyme activity by reductive methylation. A mechanism accounting for these results predicts that a (imino) covalent enzyme-substrate intermediate is formed between the protein N-terminal α-amino group and C1′ of the 5′-deoxyribose of the pyrimidine dimer substrate subsequent to (or concomitantly with) the glycosylase step. Experiments to verify the existence of this intermediate indicated that enzyme inhibition by cyanide was substrate-dependent, a result classically interpreted to imply an imino reaction intermediate. In addition, sodium borohydride reduction of the intermediate formed a stable dead-end enzyme-substrate product. This product was formed whether ultraviolet light-irradiated high molecular weight DNA or duplex oligonucleotides containing a defined thymine-thymine cyclobutane dimer were used as substrate. The duplex oligonucleotide substrates demonstrated a well-defined gel shift. This will facilitate high-resolution footprinting of the enzyme on the DNA substrate.

Original languageEnglish (US)
Pages (from-to)8284-8290
Number of pages7
JournalBiochemistry
Volume32
Issue number32
DOIs
StatePublished - Jan 1 1993

ASJC Scopus subject areas

  • Biochemistry

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