Please help with biochemistry

Question

DNA topoisomerase II unwinds DNA using the free energy derived from ATP hydrolysis to ADP and Pi. To follow continuously the catalytic turnover of DNA topoisomerse II, an enzymatic assay needs to be developed. Design an enzyme-coupled assay that involves pyruvate kinase and the cofactor NAD. Pyruvate kinase catalyzes the reversible phosphorylation of pyruvate to phosphoenolpyruvate. Also explain how you will detect the reaction and calculate enzymatic turnover rate from the coupled assay (Hint: a second enzyme is required for the coupled assay)?

Answer 1

these two resources may help shed some light to your very interesting question:

 

Stushinskaya T. et al. J. Mol Biol (2009) 385:1397-408 available on Pubmed

 

Bates et al Biochemistry (2007) 46;7929-7941

 

For a general review on topoisomerases check out chap 5 global.oup.com