Abstract
Several approaches for determining an enzyme’s kinetic parameter Km (Michaelis constant) from progress curves have been developed in recent decades. In the present article, we compare different approaches on a set of experimental measurements of lactonase activity of paraoxonase 1 (PON1): (1) a differential-equation-based Michaelis–Menten (MM) reaction model in the program Dynafit; (2) an integrated MM rate equation, based on an approximation of the Lambert W function, in the program GraphPad Prism; (3) various techniques based on initial rates; and (4) the novel program “iFIT”, based on a method that removes data points outside the area of maximum curvature from the progress curve, before analysis with the integrated MM rate equation. We concluded that the integrated MM rate equation alone does not determine kinetic parameters precisely enough; however, when coupled with a method that removes data points (e.g., iFIT), it is highly precise. The results of iFIT are comparable to the results of Dynafit and outperform those of the approach with initial rates or with fitting the entire progress curve in GraphPad Prism; however, iFIT is simpler to use and does not require inputting a reaction mechanism. Removing unnecessary points from progress curves and focusing on the area around the maximum curvature is highly advised for all researchers determining Km values from progress curves.
Highlights
Ever since Leonor Michaelis and Maud Menten first published the equation that bears their name in 1913, the Michaelis constant Km has been the main kinetic parameter used to quantify the affinity of a given enzyme for its substrate
RePON1 lactonase activity was corrected for spontaneous hydrolysis of the substrate
The present study only investigated recombinant versions of PON1 (rePON1), which is commonly used to investigate the catalytic function of paraoxonase 1 (PON1) [14,20,27]
Summary
Ever since Leonor Michaelis and Maud Menten first published the equation that bears their name in 1913, the Michaelis constant Km has been the main kinetic parameter used to quantify the affinity of a given enzyme for its substrate. An ideal method for data point selection should be based on sound mathematical principles, applicable to different types of progress curves, and capable of producing results as close as possible to real Km values. Such a method has been proposed by Stroberg and Schnell [7]. To calculate Km and Vmax with this iterative method, we selected the integrated MM equation (based on the Lambert W function) and developed a short script in Python that calculates the area of maximum curvature from a progress curve and the resulting Km and Vmax values from this area. Another advantage of rePON1 is its high stability, as reported by the researchers who developed it; G2E6 is not prone to aggregation and could be crystallized for structure determination [17]
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