EVIDENCE FOR AN IMINO INTERMEDIATE IN THE T4-ENDONUCLEASE-V REACTION

Reductive methylation and site-directed mutagenesis experiments have i mplicated the N-terminal alpha-amino group of T4 endonuclease V in the glycosylase and abasic lyase activities of the enzyme. NMR studies ha ve confirmed the involvement of the N-terminal alpha-amino group in th e inhibition of enzyme activity by reductive methylation. A mechanism accounting for these results predicts that a (imino) covalent enzyme-s ubstrate intermediate is formed between the protein N-terminal alpha-a mino group and C1' of the 5'-deoxyribose of the pyrimidine dimer subst rate subsequent to (or concomitantly with) the glycosylase step. Exper iments to verify the existence of this intermediate indicated that enz yme inhibition by cyanide was substrate-dependent, a result classicall y interpreted to imply an imino reaction intermediate. In addition, so dium borohydride reduction of the intermediate formed a stable dead-en d enzyme-substrate product. This product was formed whether ultraviole t light-irradiated high molecular weight DNA or duplex oligonucleotide s containing a defined thymine-thymine cyclobutane dimer were used as substrate. The duplex oligonucleotide substrates demonstrated a well-d efined gel shift. This will facilitate high-resolution footprinting of the enzyme on the DNA substrate.