Determination of the control coefficients allows the identification of rate-controlling steps in a reaction system. However, the measurement of the flux control coefficients in a biochemical system is not a trivial task, except for some special cases. We have developed a theoretical basis for the direct determination of these coefficients from dynamic responses. In order to show the validity of this methodology experimentally, the dynamic approach is applied to an in vitro reconstituted partial glycolytic pathway to determine the flux control coefficients of hexokinase and phosphofructokinase. It is shown that the dynamic approach gives consistent results, which agree well with values obtained by the direct enzyme titration method. The detailed procedure and potential applications to other systems, such as immobilized enzyme or cell reactors, are discussed.

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