Yeast topoisomerase II is inhibited by etoposide after hydrolyzing the first ATP and before releasing the second ADP.
Topoisomerase II-catalyzed DNA transport requires coordination between two distinct reactions: ATP hydrolysis and DNA cleavage/religation. To further understand how these reactions are coupled, inhibition by the clinically used anticancer drug etoposide was studied. The IC(50) for perturbing the DNA cleavage/religation equilibrium is nucleotide-dependent; its value is 6 microM in the presence of ATP, 25 microM in the presence of a nonhydrolyzable ATP analog, and 45 microM in the presence of ADP or no nucleotide. This inhibition was further characterized using steady-state and pre-steady-state ATPase and decatenation assays. Etoposide is a hyperbolic noncompetitive inhibitor of the ATPase activity with a K(i)(app) of 5.6 microM no inhibition of ATP hydrolysis is seen in the absence of DNA cleavage. In order to determine which steps of the ATPase mechanism etoposide inhibits, pre-steady-state analysis was performed. These results showed that etoposide does not reduce the rate of binding two ATP, hydrolyzing the first ATP, or releasing the second ADP. Inhibition is therefore associated with the first product release step or hydrolysis of the second ATP, suggesting that DNA religation normally occurs at one of these two steps. Multiple turnover decatenation is inhibited when etoposide is present; however, single turnover decatenation occurs normally. The implications of these results are discussed in terms of their contribution to our current model for the topoisomerase II mechanism.
Morris
S K
SK
Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah 84132, USA.
Lindsley
J E
JE
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D019943:Amino Acid Substitution; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D005047:Etoposide / Q000494:pharmacology*; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D059005:Topoisomerase II Inhibitors
D011994:Recombinant Proteins; D059005:Topoisomerase II Inhibitors; D000244:Adenosine Diphosphate; D005047:Etoposide; D000255:Adenosine Triphosphate; D004250:DNA Topoisomerases, Type II