Binding of non-substrate nucleotides to a restriction endonuclease: a model for the interaction of bam HI with its recognition sequence.
The kinetic constants of the site-specific endonuclease Bam HI for various substrates were determined and binding of non-substrate nucleotides to the enzyme was studied. Agarose gel assays in combination with an integrated Michaelis-Menten equation were used for the evaluation of data. The turnover number was 2.2 min-1 at 37 degrees C with pJC80 DNA as the substrate. It depends on the conformation and base composition of the substrate. Michaelis constants also depend on substrate conformation. Non-substrate polynucleotides were found to inhibit Bam competitively with KI ranging from 10(-6) to > 10(-3) M depending on base composition, base pairing, and helix conformation. Dinucleotides showed sequence-specific, competitive inhibition with KIs ranging from 10(-5) to > 10(-3) M. Mononucleotides and -nucleosides acted noncompetitively. Binding was influenced by the extent of phosphorylation, but not by the nature of the base. KIs varied between 10(-3) and 10(-2) M. The results are discussed with respect to the recognition requirements of Bam HI.
D010582:Bacteriophage lambda / Q000378:metabolism; D001435:Bacteriophages / Q000378:metabolism; D001482:Base Composition; D001483:Base Sequence; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism; D015245:Deoxyribonuclease BamHI; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000378:metabolism*; D012329:RNA, Bacterial / Q000378:metabolism; D012331:RNA, Fungal / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013329:Structure-Activity Relationship
D004279:DNA, Viral; D009711:Nucleotides; D012329:RNA, Bacterial; D012331:RNA, Fungal; D012367:RNA, Viral; D004262:DNA Restriction Enzymes; D015245:Deoxyribonuclease BamHI