The Removal of Time–Concentration Data Points from Progress Curves Improves the Determination of Km: The Example of Paraoxonase 1

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.

Vutiglabridin Modulates Paraoxonase 1 and Ameliorates Diet-Induced Obesity in Hyperlipidemic Mice

“…We found that both enantiomers of vutiglabridin significantly and dose-dependently protect against the decrease of rePON1 lactonase activity induced by OX-LA in a manner comparable to glabridin ( Figure 2 B). At 30 μM, under equivalent conditions, the rePON1 lactonase kinetic parameters K m and V max values were evaluated using iFIT program, which enables prompt and precise analysis of enzyme kinetic parameters [ 30 ]. We found that the catalytic efficiency (V max /K m ) was non-significantly increased by vutiglabridin treatment compared to the OX-LA treated group ( Table 1 ).…”

“…OX-LA was identified to specifically decrease rePON1 activity by oxidizing free sulfhydryl Cys284 and disrupting the protein stability. Glabridin was reported to bind to rePON1 at an allosteric and hydrophobic site that is different from the substrate binding site of the enzyme and modify the protein conformation to decrease the availability of Cys284, therefore protecting rePON1 from OX-LA [ 30 ]. Vutiglabridin and its enantiomers were found to bind to the same allosteric site and protect rePON1 from OX-LA-induced damage.…”

“…The protocol was generally adapted from a previous study [ 30 ]. Linoleic acid, 5,5′-dithiobis (2-nitrobenzoic acid; DTNB), 2-hydroxyquinoline, and glabridin were purchased from Sigma-Aldrich (St. Louis, MO, USA).…”

“…The catalytic activity of rePON1 was measured spectrophotometrically (SpetraMax M2, Molecular Devices, San Jose, CA, USA) at 420 nm every 2 min for 30 min. K m and V max of the kinetic graph obtained above were extracted through the iFIT program [ 30 , 31 ].…”

iFIT: An Automated Web Tool for Determining Enzyme-kinetic Parameters Based on the High-curvature Region of Progress Curves

“…4,8 At the same time, on a set of data with a purified recombinant enzyme, iFIT has been shown to compare favorably with Dynafit, the initial velocities approach, and the integrated MM equation without data point removal. 9 Using iFIT at http://www.i-fit.si (last accessed 16. February 2022), any research group that determines MM kinetic parameters from progress curves can solve the problem of unwanted data points at the beginning or end of progress curves.…”