Steady-state and rapid kinetic analysis of topoisomerase II trapped as theclosed-clamp intermediate by ICRF-193

DNA topoisomerase II uses a complex, sequential mechanism of ATP hydrolysis to catalyze the transport of one DNA duplex through a transient break in a nother. ICRF-193 is a catalytic inhibitor of topoisomerase II that is known to trap a closed-clamp intermediate form of the enzyme, Using steady-state and rapid kinetic ATPase and DNA transport assays, we have analyzed how tr apping this intermediate by the drug perturbs the topoisomerase II mechanis m. The drug has no effect on the rate of the first turnover of decatenation but potently inhibits subsequent turnovers with an IC50 of 6.5 +/- 1 mu w for the Saccharomyces cerevisiae enzyme. This drug inhibits the ATPase acti vity of topoisomerase II by an unusual, mixed-type mechanism; the drug is n ot a competitive inhibitor of ATP, and even at saturating concentrations of drug, the enzyme continues to hydrolyze ATP, albeit at a reduced rate. Top oisomerase II that was specifically isolated in the drug-bound, closed-clam p form continues to hydrolyze ATP, indicating that the enzyme clamp does no t need to re-open to bind and hydrolyze ATP, When rapid-quench ATPase assay s were initiated by the addition of ATP, the drug had no effect on the sequ ential hydrolysis of either the first or second ATP, By contrast, when the drug was prebound, the enzyme hydrolyzed one labeled ATP at the uninhibited rate but did not hydrolyze a second ATP, These results are interpreted in terms of the catalytic mechanism for topoisomerase II and suggest that ICRF -193 interacts with the enzyme bound to one ADP.