I need help with Biochem

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

An enzyme-coupled assay for DNA topoisomerase II can be designed by linking ATP hydrolysis to NADH oxidation. DNA topoisomerase II hydrolyzes ATP to ADP and Pi during its catalytic cycle. The ADP produced can be coupled to pyruvate kinase, which uses phosphoenolpyruvate (PEP) to convert ADP back into ATP while producing pyruvate. The reaction is: ADP + PEP → ATP + pyruvate. However, pyruvate formation itself is not easily followed continuously, so a second enzyme, lactate dehydrogenase, is added. Lactate dehydrogenase converts pyruvate to lactate while oxidizing NADH to NAD⁺: pyruvate + NADH + H⁺ → lactate + NAD⁺.

Since NADH absorbs strongly at 340 nm and NAD⁺ does not, the reaction can be detected by measuring the decrease in absorbance at 340 nm over time using a UV-Vis spectrophotometer. For every ATP molecule hydrolyzed by topoisomerase II, one ADP is produced, one pyruvate is formed, and one NADH molecule is oxidized. Therefore, the decrease in NADH directly reflects ATP hydrolysis by topoisomerase II.

To calculate the enzymatic turnover rate, the initial slope of the A₃₄₀ versus time curve is measured and converted to NADH concentration change using Beer-Lambert law: rate = (−dA₃₄₀/dt) / (εl), where ε₃₄₀ for NADH is 6220 M⁻¹cm⁻¹ and l is the path length. Because NADH consumption is stoichiometrically equal to ATP hydrolysis, this gives the ATP hydrolysis rate. The turnover rate is then calculated by dividing the ATP hydrolysis rate by the concentration of DNA topoisomerase II: k_cat = (ATP hydrolysis rate) / [topoisomerase II].