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 transient break in a second duplex using a complex ATP hydrolysis mechanism. Two key rates in the ATPase mechanism, ATP resynthesis and phosphate release, were investigated using 18O exchange and stopped-flow phosphate release experiments, respectively. The 18O exchange results showed that the rate of ATP resynthesis on the topoisomerase II active site was slow compared with the rate of phosphate release. When topoisomerase II was bound to DNA, phosphate was released slowly, with a lag. Since each of the preceding steps is known to occur rapidly, phosphate release is apparently a rate-determining step. The length of the lag phase was unaffected by etoposide, indicating that inhibiting DNA religation inhibits the ATPase reaction cycle at some step following phosphate release. By combining the 18O exchange and phosphate release results, the rate constant for ATP resynthesis can be calculated as approximately 0.5 s(-1). These data support the mechanism of sequential hydrolysis of two ATP by DNA topoisomerase II.
Baird
C L
CL
Department of Biochemistry and the Center for Human Toxicology, University of Utah School of Medicine, Salt Lake City, Utah 84132 and the Department of Chemistry, State University of New York, Stony Brook, New York 11794-3400.
Gordon
M S
MS
Andrenyak
D M
DM
Marecek
J F
JF
Lindsley
J E
JE
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D002851:Chromatography, High Pressure Liquid; D004250:DNA Topoisomerases, Type II / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D010710:Phosphates / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012474:Salmon; D013997:Time Factors
D005656:Fungal Proteins; D010710:Phosphates; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D004250:DNA Topoisomerases, Type II