揭示半胱胺二氧化酶的机制:HPLC-MS测定与金属置换相结合的方法。

4区 生物学 Q3 Biochemistry, Genetics and Molecular Biology
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-06-22 DOI:10.1016/bs.mie.2024.05.018
Ran Duan, Jiasong Li, Aimin Liu
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引用次数: 0

摘要

哺乳动物半胱胺二氧化酶(ADO)是一种具有三个组氨酸配体的单核非血红素铁(II)酶,在半胱胺分解代谢和调控 N-降解素信号通路中发挥着关键作用。尽管 ADO 非常重要,但其催化机理仍然难以捉摸。在这里,我们介绍了一种表征硫醇二氧合酶催化活性的 HPLC-MS 分析方法,以及一种以人类 ADO 为模型进行机理研究的金属置换方法。在氧活化方面,ADO 有两种不同的机制:一种涉及高价渡酰-氧中间体。我们假设,用一种不喜欢形成高价态的金属取代铁,可以区分不同的机制。本章详细介绍了钴取代 ADO 的表达、纯化、制备和表征。新的 HPLC-MS 分析法精确测量了酶的活性,揭示了钴取代酶中保留的反应性。研究结果表明,ADO 中同时存在二氧转移机制。这种组合方法为研究其他非血红素铁硫醇氧化酶提供了强有力的工具。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Unveiling the mechanism of cysteamine dioxygenase: A combined HPLC-MS assay and metal-substitution approach.

Mammalian cysteamine dioxygenase (ADO), a mononuclear non-heme Fe(II) enzyme with three histidine ligands, plays a key role in cysteamine catabolism and regulation of the N-degron signaling pathway. Despite its importance, the catalytic mechanism of ADO remains elusive. Here, we describe an HPLC-MS assay for characterizing thiol dioxygenase catalytic activities and a metal-substitution approach for mechanistic investigation using human ADO as a model. Two proposed mechanisms for ADO differ in oxygen activation: one involving a high-valent ferryl-oxo intermediate. We hypothesized that substituting iron with a metal that has a disfavored tendency to form high-valent states would discriminate between mechanisms. This chapter details the expression, purification, preparation, and characterization of cobalt-substituted ADO. The new HPLC-MS assay precisely measures enzymatic activity, revealing retained reactivity in the cobalt-substituted enzyme. The results obtained favor the concurrent dioxygen transfer mechanism in ADO. This combined approach provides a powerful tool for studying other non-heme iron thiol oxidizing enzymes.

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来源期刊
Methods in enzymology
Methods in enzymology 生物-生化研究方法
CiteScore
2.90
自引率
0.00%
发文量
308
审稿时长
3-6 weeks
期刊介绍: The critically acclaimed laboratory standard for almost 50 years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Each volume is eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Now with over 500 volumes the series contains much material still relevant today and is truly an essential publication for researchers in all fields of life sciences, including microbiology, biochemistry, cancer research and genetics-just to name a few. Five of the 2013 Nobel Laureates have edited or contributed to volumes of MIE.
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