Chapter 19, Problem 46P

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-phosphorus 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 ATP is isolated. If the isolated ATP is radioactive, attack occurred on the α-phosphorus. If it is not radioactive, then attack occurred on the β-phosphorus. Explain these conclusions.