How can perturbing a stationary state reveal the lifetimes of intermediates in enzyme-catalyzed reactions?

In many enzyme-catalyzed reactions, the process is experimentally irreversible, meaning it does not reach a traditional thermodynamic equilibrium. However, for much of the reaction's time course, the chemical intermediates exist in a stationary state, where their concentrations remain constant over time.1

To determine the lifetimes of these intermediates, researchers can apply a perturbation to this stationary state. By disturbing the system and measuring the rate at which the stationary state is reestablished, scientists can deduce the kinetic properties of the intermediates. This approach is particularly valuable for studying fast reactions that occur on timescales too rapid for conventional mechanical mixing techniques, which are typically limited by the millisecond-range constraints of fluid dynamics.1

In practice, these studies often utilize combined rapid mixing and relaxation techniques. By monitoring the system's response—such as through spectroscopy—researchers can observe the decay of the displacement from the stationary state. The time required for this reestablishment provides the necessary data to calculate the lifetimes and reactivities of the chemical intermediates involved in the catalytic process.12