Enzyme kinetic parameters estimation: A tricky task?

We are living in the Big Data era, and yet we may have serious troubles when dealing with a handful of kinetic data if we are not properly instructed. The aim of this paper, related to enzyme kinetics, is to illustrate how to determine the K-m and V-max of a michaelian enzyme avoiding the pitfalls in which we often fall. To this end, we will resort to kinetic data obtained by second-year Biochemistry students during a laboratory experiment using beta-galactosidase as an enzyme model, assayed at different concentrations of its substrate. When these data were analyzed using conventional linear regression of double-reciprocal plots, the range of K-m and V-max values obtained by different students varied widely. Even worse, some students obtained negative values for the kinetic parameters. Although such a scenario could make us think of a wide inter-student variability regarding their skills to obtain reliable data, the reality was quite different: when properly analyzed (accounting for error propagation) the data obtained by all the students were good enough to allow a correct estimation of the K-m (2.8 +/- 0.3 mM) and V-max (179 +/- 27 mM/min) with a reduced intergroup standard deviation. A student-accessible discussion of the importance of weighted linear regression in biochemical sciences is provided.

Automated summary

This paper discusses how to correctly determine the K-m and V-max of a michaelian enzyme, avoiding common pitfalls. Analysis of experimental data from biochemistry students revealed significant variations in results, but proper analysis could yield reliable estimates for the kinetic parameters.