Estimation of kinetic parameters, amount of endogenous substrate and contaminating enzyme activity in a target enzyme...

New analytical methods were devised to estimate the Michaelis constant (Km), the maximum velocity (V), the concentration of endogenous substrate (x) and the activity of contaminating enzyme (u) in an impure enzyme reaction with single substrate. In the non-radiometric assay, the linear plot was developed on the basis of Eisenthal-Cornish-Bowden plot [1] by transforming the equation for reaction rate (v) consisting of Km, V, x, u and the concentration of substrate (S'). To confirm the accuracy of the linear plot, the non-linear fitting method was simultaneously devised in terms of a modification of the method of Cleland [2]. In the radiometric assay, the linear and non-linear kinetic analyses were applied to the equation for the radiometric rate (v*), expressed by Km, V, x and S* (concentration of radioactively labelled substrate) as in the non-radiometric assay. In both assays, the values of Km, V and x (with u value in the non-radiometric assay) were obtainable at x1 and x2 with a known ratio of x2/x1. The validity of the above methods was proved by the model experiments with purified enzymes; and the radiometric model experiment offered a good example for a new enzymatic assay method of many substrates. These methods were successfully applied to the practical experiments.