[Determination of enzyme kinetic parameters and differentiation between various mechanisms by means of a non-linear least squares method]

Abstract

The effect produced by an inhibitor on an enzyme is characterized by the underlying mechanism and the molar inhibition coefficients Ki and Ki', respectively. The commonly used graphical estimation methods according to Lineweaver-Burk, Dixon and Cornish-Bowden do not always yield a differentiation between various possible mechanisms. According to our experience, a non-linear least-squares procedure allows a unique identification of the mechanism even in cases that are not at all, or not uniquely, solvable by graphical methods. The major advantages of this procedures and the used FORTRAN program are: 1. Fitting of the model equation itself-and not of a reciprocal conditional form-simultaneously to all measurements; 2. confidence limits for the parameter estimates; 3. residual plots to judge the goodness of fit; 4. parallel calculations and comparison of various mechanisms using the same set of data; 5. addition of further mechanisms as FORTRAN-subroutines.