Estimating the Product Inhibition Constant from Enzyme Kinetic Equations Using the Direct Linear Plot Method in One-Stage Treatment

Pedro L Valencia,Bastián Sepúlveda,Carolina Astudillo-Castro,Diego Gajardo

doi:10.3390/catal10080853

Pedro L Valencia, Bastián Sepúlveda + Show 2 more

Open Access

https://doi.org/10.3390/catal10080853

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Abstract

A direct linear plot was applied to estimate kinetic constants using the product’s competitive inhibition equation. The challenge consisted of estimating three kinetic constants, Vmax, Km, and Kp, using two independent variables, substrates, and product concentrations, in just one stage of mathematical treatment. The method consisted of combining three initial reaction rate data and avoiding the use of the same three product concentrations (otherwise, this would result in a mathematical indetermination). The direct linear plot method was highly superior to the least-squares method in terms of accuracy and robustness, even under the addition of error. The direct linear plot method is a reliable and robust method that can be applied to estimate Kp in inhibition studies in pharmaceutical and biotechnological areas.

Highlights

The direct linear plot (DLP) is a graphic method to estimate kinetic constants from enzymatic reactions based on the median as a statistic
The results indicated that DLP was applicable to equations with more than two parameters, but it was reliable and robust when compared to the least-squares (LS) method
We explored the application of DLP to the product-competitive inhibition equation, which is a different type of problem, as will be exposed

Summary

Introduction

The direct linear plot (DLP) is a graphic method to estimate kinetic constants from enzymatic reactions based on the median as a statistic. All of the recent studies regarding the application of DLP are based on the estimation of these two parameters from the Michaelis–Menten equation. The application of DLP to equations with more than two parameters has not been studied until now [3]. This median-based method was applied to estimate three kinetic constants from the substrate-uncompetitive inhibition equation. The application of DLP to estimate inhibition constants was first proposed by Eisenthal and Cornish-Bowden [1]. DLP has been used to estimate the apparent kinetic

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