Positional isotope exchange as probe of enzyme action

The positional isotope exchange technique has been found to be quite useful for the identification of reaction intermediates in enzyme-catalyzed reactions. For reactions where intermediates are not expected the method can be used with great utility for the quantitative determination of the partitioning of enzyme-product complexes. However, it must be remembered that it has been explicitly assumed that the functional group undergoing positional exchange is free to rotate. This assumption is not always valid since examples have been discovered where the functional group rotation is indeed hindered. For instance, in the reaction catalyzed by argininosuccinate synthetase a PIX reaction was not observed on incubation of ATP and citrulline even though a citrulline-adenylate complex has been identified from rapid quench experiments.