Quantitative Elucidation of Catalytic Reaction of Truncated Aldehyde Dehydrogenase Based on Linear Free Energy Relationship

IF 13.1 1区化学 Q1 CHEMISTRY, PHYSICAL ACS Catalysis Pub Date : 2025-04-18 DOI:10.1021/acscatal.4c07978

Konatsu Ichikawa, Taiki Adachi, Yuki Kitazumi, Osamu Shirai, Keisei Sowa

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Abstract

Some oxidoreductases can communicate directly and electrically with electrodes; this process is called direct electron transfer (DET)-type bioelectrocatalysis. Understanding its detailed mechanisms is essential for developing and improving DET-based bioelectrochemical devices. In this study, we investigated the pH dependence of kinetic and thermodynamic characteristics of a variant of an aldehyde dehydrogenase (ALDH) without the cytochrome c subunit (ΔC_ALDH) and compared it with that of a wild-type recombinant ALDH (rALDH). Owing to the pronounced DET activity of ΔC_ALDH at multi-walled carbon nanotubes, the voltammograms were analyzed to obtain the enzymatic parameters. The potential difference between the electrode-active site of the enzyme and electron donor (E°′_E – E°′_D) and the limiting catalytic current density (j_cat) exhibited an ideal linear free energy relationship (LFER), suggesting that the catalytic reaction of ΔC_ALDH was controlled by the thermodynamic driving force without any specific interactions. We also measured the ferricyanide reductase activity in solution (k_sol) to investigate the effect of electron acceptors (electrode and ferricyanide) on the enzymatic properties. The k_sol of ΔC_ALDH has a pH dependence similar to that of j_cat; therefore, the experimental data were kinetically analyzed based on the LFER by considering the potential difference between the electron acceptor and electrode-active site of the enzyme (E°′_A – E°′_E). By integrating the analytical results obtained from the DET-type acetaldehyde oxidation using an electrode and ferricyanide reduction in solution, the catalytic constant for the DET-type bioelectrocatalysis (k_DET) and the surface concentration of the effective enzyme immobilized on the electrode (Γ_E,eff) of ΔC_ALDH were calculated to be 5000 ± 2000 s^–1 and 13 ± 7 pmol cm^–2, respectively. This study achieved a detailed evaluation of the multi-step catalytic reactions of redox enzymes and can help elucidate the reaction mechanisms of DET-type bioelectrocatalysis.

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基于线性自由能关系的截断醛脱氢酶催化反应定量分析

一些氧化还原酶可以直接与电极电通信；这个过程被称为直接电子转移（DET）型生物电催化。了解其详细的机理对开发和改进基于det的生物电化学器件至关重要。在这项研究中，我们研究了不含细胞色素c亚基（ΔC_ALDH）的醛脱氢酶（ALDH）变体的动力学和热力学特性的pH依赖性，并将其与野生型重组ALDH （rALDH）进行了比较。由于ΔC_ALDH在多壁碳纳米管上具有明显的DET活性，通过分析其伏安图来获得酶促参数。酶的电极活性位点与电子给体的电位差（E°‘ E - E°’ D）与极限催化电流密度（jcat）呈理想的线性自由能关系（LFER），表明ΔC_ALDH的催化反应受热力学驱动力控制，没有任何特定的相互作用。我们还测量了溶液（ksol）中铁氰化物还原酶的活性，以研究电子受体（电极和铁氰化物）对酶性质的影响。ΔC_ALDH的ksol与jcat具有类似的pH依赖性；因此，考虑酶的电子受体和电极活性位点（E°‘ A - E°’ E）之间的电位差，基于LFER对实验数据进行动力学分析。通过对电极氧化和溶液中铁氰化物还原的分析结果进行综合，计算得到电极（ΓE,eff）上固定的有效酶（ΔC_ALDH）表面浓度分别为5000±2000 s-1和13±7 pmol cm-2的dt型生物电催化（kDET）催化常数。本研究对氧化还原酶的多步催化反应进行了详细的评价，有助于阐明dt型生物电催化的反应机理。

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来源期刊

ACS Catalysis CHEMISTRY, PHYSICAL-

CiteScore

20.80

自引率

6.20%

发文量

1253

审稿时长

1.5 months

期刊介绍： ACS Catalysis is an esteemed journal that publishes original research in the fields of heterogeneous catalysis, molecular catalysis, and biocatalysis. It offers broad coverage across diverse areas such as life sciences, organometallics and synthesis, photochemistry and electrochemistry, drug discovery and synthesis, materials science, environmental protection, polymer discovery and synthesis, and energy and fuels. The scope of the journal is to showcase innovative work in various aspects of catalysis. This includes new reactions and novel synthetic approaches utilizing known catalysts, the discovery or modification of new catalysts, elucidation of catalytic mechanisms through cutting-edge investigations, practical enhancements of existing processes, as well as conceptual advances in the field. Contributions to ACS Catalysis can encompass both experimental and theoretical research focused on catalytic molecules, macromolecules, and materials that exhibit catalytic turnover.