Research paperInhibiting the dimeric restriction endonuclease EcoRl using interfacial helical peptides

Add time:09/03/2019    Source:sciencedirect.com

Background: Many enzymes are active only in a dimeric form, including a variety of type II restriction endonucleases. Disruption of subunit interactions is therefore a potential method for multimeric enzyme inhibition. EcoRl is a homodimeric restriction endonuclease, the dimeric interface of which consists of a four-helix bundle. We set out to design helical peptides to interact with this interface and block dimer formation, thus rendering EcoRl inactive.Results:Here we describe two synthetic, helical peptides based on the interfacial region of EcoRl. Both peptides inhibit the enzyme, but the peptide derived from the α4 helix of EcoRl had both a higher helical content and better efficacy than a variant peptide, α4(Leu), that has three Ile→Leu mutations (IC50 values of 27 μM and 90 μM, and helical contents of 29% and 10%, respectively). Size-exclusion chromatography confirmed that the α4 peptide disrupted dimerization of EcoRl, and circular dichroism indicated that EcoRl remained folded upon binding to a4. Inhibition with α4 and α4(Leu) was shown to be specific for EcoRl, as the dimeric restriction enzyme Pvull was not affected by the peptides.Conclusions: Interfacial peptide inhibitors of the dimeric EcoRl were obtained that both inhibit dimerization and endonuclease activity. The peptide sequence with a preference for a helical conformation was a more effective inhibitor, presumably because the more preorganized state enhanced interactions with the helical interface of EcoRl. The specific nature of this endonuclease-peptide interaction was also confirmed. The potential of this strategy for inhibiting other enzyme classes is currently being addressed.

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