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 en zyme titration method in which the change in pathway flux upon a chang e in the enzyme concentration is measured. In this study, the applicat ion 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 mathematica l construct 'steady state'. It was shown that flux control coefficient s, calculated in a way that mimics their experimental determination, w ere strongly time dependent. Initially, the calculated flux control co efficient 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 fro m the monitoring reaction. The observed time course of simulated flux control coefficients was shown to reflect the fact that experimental s ystems are not steady state but quasi-steady-state. For a pathway in t he quasi-steady-state, some of the problems with enzyme titration expe riments can be overcome by allowing the system to relax for a time int erval that is large compared with the turnover time of the pooled path way intermediates.