Mechanism of inhibition of vaccinia DNA topoisomerase by novobiocin and coumermycin.
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 K1 values of 10-25 microM for coumermycin and 350 microM for novobiocin. Spectroscopic and fluorescence measurements of drug binding t enzyme indicate a single binding site on vaccinia topoisomerase for coumermycin (KD = 27 +/- 5 microM) and two classes of binding sites for novobiocin, one tight site (KD1 = 20 +/- 5 microM) and several weak sites (KD2 = 513 +/- 125 microM; n = 4.9 +/- 0.7). Addition of a stoichiometric amount of DNA to a performed 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.
Sekiguchi
J
J
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
Stivers
J T
JT
Mildvan
A S
AS
Shuman
S
S
D049933:Aminocoumarins; D001483:Base Sequence; D001667:Binding, Competitive; D003374:Coumarins / Q000494:pharmacology; D004278:DNA, Superhelical / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007700:Kinetics; D008969:Molecular Sequence Data; D009675:Novobiocin / Q000494:pharmacology*; D010449:Peptide Mapping; D011485:Protein Binding; D059004:Topoisomerase I Inhibitors; D014616:Vaccinia virus / Q000201:enzymology*
D049933:Aminocoumarins; D003374:Coumarins; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D059004:Topoisomerase I Inhibitors; D009675:Novobiocin; C004628:coumermycin