Evidence for an imino intermediate in the T4 endonuclease V reaction

M. L. Dodson, Robert (Stephen) Lloyd, R. S. Lloyd

Research output: Contribution to journalArticle

132 Citations (Scopus)

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
StatePublished - 1993
Externally publishedYes

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Substrates
Enzymes
Pyrimidine Dimers
Enzyme inhibition
Methylation
Oligonucleotides
Deoxyribose
Lyases
DNA
Cyanides
Reaction intermediates
Mutagenesis
Ultraviolet Rays
Site-Directed Mutagenesis
Enzyme activity
phage T4 endonuclease V
Molecular Weight
Gels
Experiments
Molecular weight

ASJC Scopus subject areas

  • Biochemistry

Cite this

Evidence for an imino intermediate in the T4 endonuclease V reaction. / Dodson, M. L.; Lloyd, Robert (Stephen); Lloyd, R. S.

In: Biochemistry, Vol. 32, No. 32, 1993, p. 8284-8290.

Research output: Contribution to journalArticle

Dodson, ML, Lloyd, RS & Lloyd, RS 1993, 'Evidence for an imino intermediate in the T4 endonuclease V reaction', Biochemistry, vol. 32, no. 32, pp. 8284-8290.
Dodson, M. L. ; Lloyd, Robert (Stephen) ; Lloyd, R. S. / Evidence for an imino intermediate in the T4 endonuclease V reaction. In: Biochemistry. 1993 ; Vol. 32, No. 32. pp. 8284-8290.
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