Determination of the kinetic properties of enzymes catalysing coupled reaction sequences

In a coupled system of two enzyme-catalysed reactions the concentration of the final product follows a sigmoidal progress curve. The shape of this curve is dependent on the concentrations and kinetic properties of both enzymes in the system; provided that the activities of the two enzymes are similar, the kinetic properties of both enzymes may be determined simultaneously by analysis of such curves. The value of this approach is substantiated using a model system consisting of pyruvate kinase (EC 2.7.1.40) and lactate dehydrogenase (EC 1.1.1.27). Analysis of the data by a combination of numerical integration and nonlinear regression yielded values for the kinetic parameters which are in excellent agreement with those determined from measurements of steady-state rates. Appropriate experimental designs for this type of progress curve analysis are considered. The analysis is compared with, and is shown to be of much greater generality than, published procedures involving measurements of the early stages of progress curves.