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 language | English (US) |
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Pages (from-to) | 8284-8290 |
Number of pages | 7 |
Journal | Biochemistry |
Volume | 32 |
Issue number | 32 |
DOIs | |
State | Published - 1993 |
Externally published | Yes |
ASJC Scopus subject areas
- Biochemistry