Evidence for an imino intermediate in the T4 endonuclease V reaction.
Reductive methylation and site-directed mutagenesis experiments have implicated the N-terminal alpha-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 alpha-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 alpha-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.
Dodson
M L
ML
Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston 77555.
Schrock
R D
RD
Lloyd
R S
RS
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001894:Borohydrides / Q000494:pharmacology; D002417:Cattle; D003488:Cyanogen Bromide / Q000494:pharmacology; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage; D004260:DNA Repair; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007097:Imines / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008745:Methylation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011740:Pyrimidine Dimers / Q000378:metabolism; D013329:Structure-Activity Relationship; D014466:Ultraviolet Rays; D014764:Viral Proteins
D001894:Borohydrides; D007097:Imines; D011740:Pyrimidine Dimers; D014764:Viral Proteins; C025364:sodium borohydride; D004247:DNA; D004706:Endodeoxyribonucleases; C470698:endonuclease V, phage T4; D043224:Deoxyribonuclease (Pyrimidine Dimer); D003488:Cyanogen Bromide