Mechanism of inhibition of vaccinia DNA topoisomerase by novobiocin and coumermycin Journal Article
| Authors: | Sekiguchi, J.; Stivers, J. T.; Mildvan, A. S.; Shuman, S. |
| Article Title: | Mechanism of inhibition of vaccinia DNA topoisomerase by novobiocin and coumermycin |
| Abstract: | Vaccinia DNA topoisomerase, a eukaryotic type I enzyme, has unique pharmacological properties, including sensitivity to the coumarin drugs novobiocin and coumermycin, which are classical inhibitors of DNA gyrase, a type II enzyme. Whereas coumarins inhibit gyrase by binding the GyrB subunit and thereby blocking the ATP-binding site, they inhibit vaccinia topoisomerase by binding to the protein and blocking the interaction of enzyme with DNA. Noncovalent DNA binding and single-turnover DNA cleavage by topoisomerase are inhibited with K(I) values of 10-25 μM for coumermycin and 350 μM for novobiocin. Spectroscopic and fluorescence measurements of drug binding to enzyme indicate a single binding site on vaccinia topoisomerase for coumermycin (K(D) = 27 ± 5 μM) and two classes of binding sites for novobiocin, one tight site (K(D1) = 20 ± 5 μM) and several weak sites (K(D2) = 513 ± 125 μM; n = 4.9 ± 0.7). Addition of a stoichiometric amount of DNA to a preformed coumermycin-topoisomerase complex quantitatively displaces the drug, indicating that coumermycin binding and DNA binding to topoisomerase are mutually exclusive. A simple interpretation is that the site of drug binding coincides or overlaps with the DNA-binding site on the topoisomerase. Both novobiocin and coumermycin alter the susceptibility of vaccinia topoisomerase to proteolysis with either chymotrypsin or trypsin; similar effects occur when topoisomerase binds to duplex DNA. |
| Keywords: | controlled study; nonhuman; protein degradation; fluorescence; protein binding; dna; eukaryota; vaccinia virus; binding site; adenosine triphosphate; dna topoisomerase (atp hydrolysing); dna binding; spectroscopy; chymotrypsin; drug binding; dna cleavage; dna topoisomerase; trypsin; vaccinia; gel electrophoresis; novobiocin; coumarin derivative; coumamycin a1; priority journal; article |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 271 |
| Issue: | 4 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 1996-01-26 |
| Start Page: | 2313 |
| End Page: | 2322 |
| Language: | English |
| DOI: | 10.1074/jbc.271.4.2313 |
| PROVIDER: | scopus |
| PUBMED: | 8567695 |
| DOI/URL: | |
| Notes: | Article -- Export Date: 22 November 2017 -- Source: Scopus |
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