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 enzy...

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Bibliographic Details
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
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Summary: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.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi00083a032