Nernst-Michaelis-Menten framework unlocks electrochemical kinetics for laccases

Abstract

Determining oxidoreductase kinetic parameters remains challenging due to spectrophotometric method limitations. Here, we present an innovative approach combining electrochemistry and enzymology principles through a novel Nernst-Michaelis-Menten theoretical framework. This model merges the Nernst equation, describing electrochemical equilibrium, with Michaelis-Menten kinetics, enabling accurate enzyme parameters determination, via chronopotentiometry. Using a commercial laccase from Trametes versicolor as a model system, we demonstrate precise kinetic parameters measurement for both chromophoric (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) - ABTS, K_m = (56.7 ± 6.3) μM) and non-chromophoric (hydroquinone, K_m = (196 ± 59) μM) substrates, validated against established techniques. The method requires minimal enzyme quantities and enables rapid analysis. This approach overcomes current methodological limitations and extends to other oxidoreductases, providing a powerful tool for enzyme characterization. Our work provides a new paradigm for enzyme kinetics, expanding the scope of analysable enzymatic systems, including those that were previously challenging to characterize with conventional methods.

Keywords: Chronopotentiometry; Enzyme kinetics; Laccase; Oxidase; Redox potential.