Concerning the measurement of flux control coefficients by enzyme titration. Steady states, quasi-steady-states, and the role of time in control analytical experiments.

An established method to determine flux control coefficients is the enzyme titration method in which the change in pathway flux upon a change in the enzyme concentration is measured. In this study, the application of this method to a simple reconstituted pathway was investigated by simulated measurements. The pathway was assumed to be in the quasi-steady-state, which is the experimental realization of the mathematical construct 'steady state'. It was shown that flux control coefficients, calculated in a way that mimics their experimental determination, were strongly time dependent. Initially, the calculated flux control coefficient was high for the enzyme adjacent to the reaction monitoring the flux, and the steady-state value was overestimated. Likewise, flux control coefficients were underestimated for enzymes further away from the monitoring reaction. The observed time course of simulated flux control coefficients was shown to reflect the fact that experimental systems are not steady state but quasi-steady-state. For a pathway in the quasi-steady-state, some of the problems with enzyme titration experiments can be overcome by allowing the system to relax for a time interval that is large compared with the turnover time of the pooled pathway intermediates.