Chapter 24, Problem 53P

A student is trying to determine the mechanism for a reaction that uses ATP to activate a carboxylate ion, which then reacts with a thiol. If the carboxylate ion attacks the γ-phosphorous of ATP, the reaction products are the thioester, ADP, and phosphate. However, whether it attacks the α-phosphorus or the β-phosphorus of ATP cannot be determined from the reaction products because the thioester, AMP, and pyrophosphate would be the products in both reactions. The mechanisms can be distinguished by a labeling experiment in which the enzyme, the carboxylate ion, ATP, and radioactively labeled pyrophosphate are incubated and then the ATP is isolated. If the isolated ATP is radioactive, attack occurred on the α-phosphorous. If it is not radioactive, then attack occurred on the β-phosphorous. Explain these conclusions.