Energy coupling in DNA gyrase and the mechanism of action of novobiocin

Abstract

Escherichia coli DNA gyrase catalyzes negative supercoiling of closed duplex DNA at the expense of ATP. Two additional activities of the enzyme that illuminated the energy coupling component of the supercoiling reaction are the DNA-dependent hydrolysis of ATP to ADP and Pi and the alteration by ATP of the DNA site specificity of the gyrase cleavage reaction. This cleavage of both DNA strands results from treatment with sodium dodecyl sulfate of the stable gyrase-DNA complex that is trapped by the inhibitor oxolinic acid. Either ATP or a nonhydrolyzable analog, adenyl-5''-yl-imidodiphosphate (App[NH]p), shifts the primary cleavage site on ColE1 DNA. The prevention by novobiocin and coumermycin A1 of this cleavage rearrangement places the site of action of the antibiotics at a reaction step prior to ATP hydrolysis. The step blocked is the binding of ATP because coumermycin A1 and novobiocin interact competitively with ATP in the ATPase and supercoiling assays; the Ki [inhibition constant] values are more than 4 orders of magnitude less than the Km for ATP. This simple mechanism accounts for all effects of the drugs on DNA gyrase. Studies with App[NH]p, another potent competitive inhibitor of reactions catalyzed by gyrase, show that cleavage of a high energy bond is not required for driving DNA into the higher energy supercoiled form. With substrate levels of gyrase, App[NH]p induces supercoiling that is proportional to the amount of enzyme; a -0.3 superhelical turn was introduced/gyrase protomer A. ATP and App[NH]p are apparently allosteric effectors of a conformational change of gyrase that leads to 1 round of supercoiling. Nucleotide dissociation favored by hydrolysis of ATP returns gyrase to its original conformation and thereby permits enzyme turnover. Such cyclic conformational changes accompanying alteration in nucleotide affinity also seem to be a common feature of energy transduction in other diverse processes including muscle contracton, protein synthesis and oxidative phosphorylation.