ANALYSIS OF PROGRESS CURVES FOR ENZYME-CATALYZED REACTIONS - APPLICATION TO UNSTABLE ENZYMES, COUPLED REACTIONS AND TRANSIENT-STATE KINETICS

There are several advantages to the use of progress curves to analyze the kinetic properties of enzymes but most studies still rely on rate measurements. One of the reasons for this may be that progress curve a nalysis relies on the enzyme and the reactants being completely stable under assay conditions. Here a method is described that relaxes this requirement and allows progress curve analysis to be applied to unstab le enzymes. The procedure is based on a combination of numerical integ ration and non-linear regression to fit rate equations to the progress curve data. The analysis is verified using simulated data and illustr ated by application to the reaction catalyzed by alkaline phosphatase, measured in the presence of 10 mM EGTA where it has a half-life of 31 /2 min. The method may also be applied to other experimental systems w here the development over time reveals important properties but where an analytical solution of the underlying model is not known. This exte nsion is illustrated by two systems: the coupled reactions catalyzed b y pyruvate kinase and lactate dehydrogenase under conditions where bot h enzymes have similar activity; and the transient-state kinetics of t he reaction catalyzed by glutamate dehydrogenase.