When multiple recognition sites with different flanking sequences are present, most restriction enzymes show a slight preference and cleave these sites at different rates. These rate differences make it possible that adding a small excess of enzyme will avoid any problems caused by incomplete digestion. However, as always, one must be aware of the experimental molar concentration of recognition sites and the digestion conditions associated with the definition of the unit. Creative Enzymes provides you with a variety of site preferences research services t to meet your scientific research needs. This service is aimed at developers who need drug level quantification and enzyme kinetics for therapeutic enzymes, enzyme inhibitors or enzyme and protein researchers.
Some restriction enzymes have considerable difficulty in cleaving some of their recognition sites. Original experiments using these enzymes resulted in specifying their site preferences, as follows:
| Cleavable sites | >90% cleavage with 1-5 fold excess enzyme |
| Slow sites | 5-90% cleavage with 1-5 fold excess and additional cleavage with 10-30 fold excess |
| Resistant sites | <5% cleavage with 5 fold excess enzyme and no additional cleavage with 10-30 fold excess |
Notes: Enzymes with cleavable, slow and resistant sites in the same or different DNA have been designated as type IIe restriction enzymes. This group consists of enzymes that are otherwise members of the common type II or IIs classes. type IIe enzymes are NaeI, NarI, BspMI, HpaII, SacII, EcoRII, AtuBI, Crf9I, SauBMKI, and Ksp632I.
Investigations have shown that a second recognition sequence binds to the distal non-catalytic site on the enzyme in either a cis or trans manner and can render the slow and resistant site cleavable. This effector sequence alters the kinetics in one of two ways. In the K class (NarI, HpaII, SacII), activator DNA binding decreases K m without altering the cleaved V max, suggesting that concerted binding induces a conformational shift that increases the affinity of the enzyme for its substrate. It is hypothesized that the flanking sequence of the recognition site affects the kinetics of cleavage at the site, but this interaction is not known at this time. There is also considerable variation in the ability of effector sequences to stimulate cleavage. Recognition sites flanked by sequences from sites that are susceptible to cleavage are a useful starting point for designing good effector sequences.
Creative Enzymes is a company that provides professional and comprehensive site preferences research services t. We have years of experience to meet your specific project needs in using enzyme research to add value to your research projects. Creative Enzymes can provide you with personalized solutions to help you thrive every step of the way around your interest in your workflow. If you would like to know more about this service, please feel free to contact us.
We are here to answer any question you may have
