| Abstract
| - In the preceding paper, we showed that DNAtopoisomerase II from Saccharomyces cerevisiaebinds two ATP and rapidly hydrolyzes at least one of them beforeencountering a slow step in the reactionmechanism. These data are potentially consistent with twodifferent types of reaction pathways: (1)sequential ATP hydrolysis or (2) simultaneous hydrolysis of both ATP.Here, we present results that areconsistent only with topoisomerase II hydrolyzing its two bound ATPsequentially. Additionally, theseresults indicate that the products of the first hydrolysis are releasedfrom the enzyme before the secondATP is hydrolyzed. Release of products from both the first andsecond hydrolyses contributes to therate-determining process. The proposed mechanism for ATPhydrolysis by topoisomerase II is complex,having nine rate constants. To calculate values for each of theserate constants, a technique of kineticparameter estimation was developed. This technique involved usingsingular perturbation theory in orderto estimate rate constants, and consequently identify kinetic stepsfollowing the rate-determining step.
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