Evidence for an imino intermediate in the T4 endonuclease V reaction

• Published in: Biochemistry (Easton) 1993-08, Vol.32 (32), p.8284-8290
• Main Authors: Dodson, M. L, Schrock, Robert D, Lloyd, R. Stephen
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Base Sequence; Binding Sites; Biological and medical sciences; Borohydrides - pharmacology; Cattle; Cyanogen Bromide - pharmacology; Deoxyribonuclease (Pyrimidine Dimer); DNA - metabolism; DNA Damage; DNA Repair; Endodeoxyribonucleases - chemistry; Endodeoxyribonucleases - genetics; Endodeoxyribonucleases - metabolism; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Hydrolases; Imines - metabolism; Magnetic Resonance Spectroscopy; Methylation; Molecular Sequence Data; Mutagenesis, Site-Directed; phage T4; Pyrimidine Dimers - metabolism; Structure-Activity Relationship; Ultraviolet Rays; Viral Proteins
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00083a032

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.