The ATPase reaction cycle of yeast DNA topoisomerase II - Slow rates of ATP resynthesis and P-i release

DNA topoisomerase II catalyzes the transport of one DNA duplex through a tr ansient break in a second duplex using a complex ATP hydrolysis mechanism. Two key rates in the ATPase mechanism, ATP resynthesis and phosphate releas e, were investigated using O-18 exchange and stopped-flow phosphate release experiments, respectively. The O-18 exchange results showed that the rate of ATP resynthesis on the topoisomerase II active site was slow compared wi th the rate of phosphate release. When topoisomerase II was bound to DNA, p hosphate was released slowly, with a lag. Since each of the preceding steps is known to occur rapidly, phosphate release is apparently a rate-determin ing step. The length of the lag phase was unaffected by etoposide, indicati ng that inhibiting DNA religation inhibits the ATPase reaction cycle at som e step following phosphate release. By combining the O-18 exchange and phos phate release results, the rate constant for ATP resynthesis can be calcula ted as similar to0.5 s(-1). These data support the mechanism of sequential hydrolysis of two ATP by DNA topoisomerase II.