3D Kinetic Analysis: A Rapid, Model Independent, Method for Enzyme Kinetics
Research Square (Research Square)(2021)
Abstract
Abstract In this work, we present the 3D Kinetics approach as a step forward in the field of enzyme kinetics. Normally in enzyme kinetics, it is first assumed that the kinetics will conform to the assumption of Michaelis and Menten and an experiment is conducted at various concentrations around the concentration that gives half-maximal velocity. Often, these experiments could be compromised by having too much substrate, nonlinear reaction over time, time-dependent or substrate inhibition, or several other kinetic models. Herein, we present a general strategy that will decrease the number of experiments required to develop an accurate representation of the kinetics of an enzymatic reaction. We show that with a single experimental protocol, we can fit a number of the most common kinetic models associated with enzyme-catalyzed reactions. Through this experiment, we introduce the effect of time on saturation curves by modeling the reaction velocity over time and across a set of substrate concentrations. Michaelis-Menten (MM) kinetics and other analytical solutions used to solve more complex kinetic models were introduced to the field of enzymology over a hundred years ago and have only marginally changed over the years with each analytical model requiring a different set of experiments and concentration ranges. Although this approach was necessary at the time, the computational power today makes any such simplifying and limiting efforts unnecessary and avoidable. In this study, we use a single experimental protocol and fit a number of different models to the resulting data. We present four different case studies to compare and contrast the outcomes of 3D Kinetics with MM analysis for different kinetic scenarios such as enzymatic reactions with linear kinetics, biphasic kinetics, substrate inhibition, and time-dependent inhibition (TDI) to confirm the advantage of the 3D Kinetics method to the long-established MM analysis.
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Key words
3d kinetic analysis,enzyme,kinetics
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