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

Abstract

New analytical methods were devised to estimate the Michaelis constant (K_m), 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 K_m, 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 ($\text{v}{}^{\mathrm{\ast }}$), expressed by K_m, V, x and $\text{S}{}^{\mathrm{\ast }}$ (concentration of radioactively labelled substrate) as in the non-radiometric assay. In both assays, the values of K_m, 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.