Nernst-Michaelis-Menten framework unlocks electrochemical kinetics for laccases

IF 4.8 2区化学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Bioelectrochemistry Pub Date : 2025-05-13 DOI:10.1016/j.bioelechem.2025.109003

Elise Martin , Fabrice Audonnet , Daniel Yaacoub , Pascal Dubessay , Philippe Michaud

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引用次数: 0

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.

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Nernst-Michaelis-Menten框架揭示了漆酶的电化学动力学

由于分光光度法的局限性，测定氧化还原酶动力学参数仍然具有挑战性。在这里，我们提出了一种结合电化学和酶学原理的创新方法，通过一个新的能斯特-米切里斯-门腾理论框架。该模型将描述电化学平衡的能斯特方程与米切利斯-门腾动力学相结合，通过时间电位测定法精确测定酶的参数。使用来自Trametes versicolor的商业漆酶作为模型系统，我们展示了对显色性（2,2 ' -氮基-双（3-乙基苯并噻唑啉-6-磺酸）- ABTS, Km =(56.7±6.3)μM）和非显色性（对苯二酚，Km =(196±59)μM）底物的精确动力学参数测量，并根据现有技术进行了验证。该方法需要最少的酶量，能够快速分析。该方法克服了目前方法的局限性，并扩展到其他氧化还原酶，为酶表征提供了有力的工具。我们的工作为酶动力学提供了一个新的范例，扩大了可分析酶系统的范围，包括那些以前难以用传统方法表征的系统。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Bioelectrochemistry 生物-电化学

CiteScore

9.10

自引率

6.00%

发文量

238

审稿时长

38 days

期刊介绍： An International Journal Devoted to Electrochemical Aspects of Biology and Biological Aspects of Electrochemistry Bioelectrochemistry is an international journal devoted to electrochemical principles in biology and biological aspects of electrochemistry. It publishes experimental and theoretical papers dealing with the electrochemical aspects of: • Electrified interfaces (electric double layers, adsorption, electron transfer, protein electrochemistry, basic principles of biosensors, biosensor interfaces and bio-nanosensor design and construction. • Electric and magnetic field effects (field-dependent processes, field interactions with molecules, intramolecular field effects, sensory systems for electric and magnetic fields, molecular and cellular mechanisms) • Bioenergetics and signal transduction (energy conversion, photosynthetic and visual membranes) • Biomembranes and model membranes (thermodynamics and mechanics, membrane transport, electroporation, fusion and insertion) • Electrochemical applications in medicine and biotechnology (drug delivery and gene transfer to cells and tissues, iontophoresis, skin electroporation, injury and repair). • Organization and use of arrays in-vitro and in-vivo, including as part of feedback control. • Electrochemical interrogation of biofilms as generated by microorganisms and tissue reaction associated with medical implants.