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The removal of time–concentration data points from progress curves improves the determination of K$_m$ : the example of paraoxonase 1
ID
Petrič, Boštjan
(
Author
),
ID
Goličnik, Marko
(
Author
),
ID
Bavec, Aljoša
(
Author
)
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https://www.mdpi.com/1420-3049/27/4/1306
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Abstract
Several approaches for determining an enzyme’s kinetic parameter K$_m$ (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 K$_m$ values from progress curves.
Language:
English
Keywords:
paraoxonase 1
,
dihydrocoumarin
,
progress curves
,
integrated Michaelis–Menten equation
,
Lambert W
,
lactonase activity
Work type:
Article
Typology:
1.01 - Original Scientific Article
Organization:
MF - Faculty of Medicine
Publication status:
Published
Publication version:
Version of Record
Year:
2022
Number of pages:
17 str.
Numbering:
Vol. 27, iss. 4, art. 1306
PID:
20.500.12556/RUL-137296
UDC:
577
ISSN on article:
1420-3049
DOI:
10.3390/molecules27041306
COBISS.SI-ID:
98612227
Publication date in RUL:
09.06.2022
Views:
772
Downloads:
104
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Title:
Molecules
Shortened title:
Molecules
Publisher:
MDPI
ISSN:
1420-3049
COBISS.SI-ID:
18462981
Licences
License:
CC BY 4.0, Creative Commons Attribution 4.0 International
Link:
http://creativecommons.org/licenses/by/4.0/
Description:
This is the standard Creative Commons license that gives others maximum freedom to do what they want with the work as long as they credit the author.
Licensing start date:
15.02.2022
Secondary language
Language:
Slovenian
Keywords:
paraoksonaza 1
,
dihidrokumarin
,
krivulje napredka
Projects
Funder:
ARRS - Slovenian Research Agency
Project number:
P1-0170
Name:
Molekulski mehanizmi uravnavanja celičnih procesov v povezavi z nekaterimi boleznimi pri človeku
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