The removal of time–concentration data points from progress curves improves the determination of K$_m$ : the example of paraoxonase 1

Petrič, Boštjan (avtor), Goličnik, Marko (avtor), Bavec, Aljoša (avtor)

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.

• Jezik: Angleški jezik
• Ključne besede: paraoxonase 1, dihydrocoumarin, progress curves, integrated Michaelis–Menten equation, Lambert W, lactonase activity
• Vrsta gradiva: Članek v reviji
• Tipologija: 1.01 - Izvirni znanstveni članek
• Organizacija: MF - Medicinska fakulteta
• Status publikacije: Objavljeno
• Različica publikacije: Objavljena publikacija
• Leto izida: 2022
• Št. strani: 17 str.
• Številčenje: Vol. 27, iss. 4, art. 1306
• PID: 20.500.12556/RUL-137296
• UDK: 577
• ISSN pri članku: 1420-3049
• DOI: 10.3390/molecules27041306
• COBISS.SI-ID: 98612227
• Datum objave v RUL: 09.06.2022

Gradivo je del revije

• Naslov: Molecules
• Skrajšan naslov: Molecules
• Založnik: MDPI
• ISSN: 1420-3049
• COBISS.SI-ID: 18462981

Licence

• Licenca: CC BY 4.0, Creative Commons Priznanje avtorstva 4.0 Mednarodna
• Povezava: http://creativecommons.org/licenses/by/4.0/deed.sl
• Opis: To je standardna licenca Creative Commons, ki daje uporabnikom največ možnosti za nadaljnjo uporabo dela, pri čemer morajo navesti avtorja.
• Začetek licenciranja: 15.02.2022

Sekundarni jezik

• Jezik: Slovenski jezik
• Ključne besede: paraoksonaza 1, dihidrokumarin, krivulje napredka

Projekti

• Financer: ARRS - Agencija za raziskovalno dejavnost Republike Slovenije
• Številka projekta: P1-0170
• Naslov: Molekulski mehanizmi uravnavanja celičnih procesov v povezavi z nekaterimi boleznimi pri človeku