Mechanism of inhibition of vaccinia DNA topoisomerase by novobiocin and coumermycin

Joann Sekiguchi, James Stivers, Albert S. Mildvan, Stewart Shuman

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)2313-2322
Number of pages10
JournalJournal of Biological Chemistry
Volume271
Issue number4
DOIs
StatePublished - Jan 26 1996

Fingerprint

Novobiocin
Vaccinia
Type I DNA Topoisomerase
Binding Sites
DNA
Enzymes
Pharmaceutical Preparations
Topoisomerase II Inhibitors
Coumarins
Proteolysis
Chymotrypsin
coumermycin
Carrier Proteins
Trypsin
Adenosine Triphosphate
Fluorescence
Pharmacology

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mechanism of inhibition of vaccinia DNA topoisomerase by novobiocin and coumermycin. / Sekiguchi, Joann; Stivers, James; Mildvan, Albert S.; Shuman, Stewart.

In: Journal of Biological Chemistry, Vol. 271, No. 4, 26.01.1996, p. 2313-2322.

Research output: Contribution to journalArticle

Sekiguchi, Joann ; Stivers, James ; Mildvan, Albert S. ; Shuman, Stewart. / Mechanism of inhibition of vaccinia DNA topoisomerase by novobiocin and coumermycin. In: Journal of Biological Chemistry. 1996 ; Vol. 271, No. 4. pp. 2313-2322.
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