Differences in the kinetic properties of BamHI endonuclease and methylase with linear DNA substrates.
BamHI endonuclease and methylase were found to exhibit a kinetic preference for linear pBR322 DNA substrates containing the recognition site in a central location. The Km values for substrates having the recognition site in a terminal location were approximately 3-fold greater than those with a centrally located site. This phenomenon may be partially due to facilitated transfer of the enzymes to the recognition site over nonspecific flanking sequences. The exploitation of facilitated transfer by these enzymes has been inferred from studies demonstrating kinetic preferences for longer DNA substrates. The reaction rates of the endonuclease were 9-fold greater with full-length pBR322 DNA than with a 74-base pair derivative. The methylase exhibits a kinetic preference for longer substrates but only under conditions of comparatively higher DNA concentrations. In addition, the methylase has the property of increasing long chain preference with increasing salt concentrations up to 120 mM. Increasing salt concentrations decreased the endonuclease's preference for longer substrates. Nonspecific inhibition studies revealed qualitative and quantitative differences between the two enzymes under catalytic conditions. These studies suggest that BamHI endonuclease and methylase interact with nonspecific DNA in different ways.
D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism*; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism*; D015245:Deoxyribonuclease BamHI; D015246:Deoxyribonuclease EcoRI; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics
D004269:DNA, Bacterial; D004248:DNA (Cytosine-5-)-Methyltransferases; D004262:DNA Restriction Enzymes; D015245:Deoxyribonuclease BamHI; D015246:Deoxyribonuclease EcoRI