ENZYPLOTW: A USEFUL TOOL TO CALCULATE ENZYME KINETIC PARAMETERS

Abstract

Assays of enzyme activity are among the most frequently employed procedures in biochemistry. They are used to estimate the amount of a given enzyme in a cell or tissue, to quantify kinetic parameters or to investigate a catalytic mechanism. The purpose of enzyme kinetics obtained under steady-state conditions is to estimate KM and VM values by fitting initial rate (v) and substrate concentration (S) values to the Michaelis-Menten equation, permitting a convenient graphical representation and an accurate estimation of KM and VM. However, there is no ideal assay for any particular enzyme and, since the assay of enzyme activity is essentially a kinetic measurement, various pitfalls await the unwary investigator. Despite difficulties consequent to intrinsic enzyme properties, kinetic parameters can be estimated accurately when steady-state conditions (<10% variation in substrate concentration during activity assay) and initial rate measurements (catalytic enzyme concentration) are guaranteed.EnzyplotW is an application developed for use on any Intel-based computer running Windows 98 or later, and uses a non-linear regression method to fit steady-state kinetic data for Michaelian enzymes. EnzyplotW can be employed not only for educational purposes but also in routine laboratory work, and includes three important features: teaching students how to obtain and recognize sound data for the best estimation of kinetic parameter values; aid the investigator at the bench in gauging the limitations of the various equivalent plots of the Michaelis-Menten equation used to estimate KM and VM; and allow the student to manipulate the mathematical formula of the corresponding plot to comprehend the qualitative and quantitative issues that govern the relationship between substrate concentration and reaction rate.