Molecular Design and Synthesis of a Novel Substrate for Assaying Lysozyme Activity.

IF 16.4 1区化学 Q1 CHEMISTRY, MULTIDISCIPLINARY

Accounts of Chemical Research Pub Date : 2018-08-20 eCollection Date: 2018-01-01 DOI:10.5458/jag.jag.JAG-2018_003

Megumi Matsui, Haruka Kono, Makoto Ogata

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Abstract

A novel substrate {Galβ1,4GlcNAcβ1,4GlcNAc-β-pNP [Gal(GlcNAc)₂-β-pNP]} for assaying lysozyme activity has been designed using docking simulations and enzymatic synthesis via β-1,4-galactosyltransferase-mediated transglycosylation from UDP-Gal as the donor to (GlcNAc)₂-β-pNP as the acceptor. Hydrolysis of the synthesized Gal(GlcNAc)₂-β-pNP and related compounds using hen egg-white lysozyme (HEWL) demonstrated that the substrate was specifically cleaved to Gal(GlcNAc)₂ and p-nitrophenol (pNP). A combination of kinetic studies and docking simulation was further conducted to elucidate the mode of substrate binding. The results demonstrate that Gal(GlcNAc)₂-β-pNP selectively binds to a subsite of lysozyme to liberate the Gal(GlcNAc)₂ and pNP products. The work therefore describes a new colorimetric method for quantifying lysozyme on the basis of the determination of pNP liberated from the substrate.

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检测溶菌酶活性的新型底物的分子设计与合成

通过对接模拟设计了一种用于检测溶菌酶活性的新型底物 {Galβ1,4GlcNAcβ1,4GlcNAc-β-pNP [Gal(GlcNAc)2-β-pNP]}，并通过β-1,4-半乳糖基转移酶介导的转糖基化将 UDP-Gal 作为供体与 (GlcNAc)2-β-pNP 作为受体进行酶法合成。用母鸡卵白溶菌酶（HEWL）水解合成的Gal（GlcNAc）2-β-pNP和相关化合物的结果表明，底物被特异性地裂解为Gal（GlcNAc）2和对硝基苯酚（pNP）。为了阐明底物的结合模式，研究人员进一步将动力学研究和对接模拟相结合。结果表明，Gal(GlcNAc)2-β-pNP 可选择性地与溶菌酶的一个位点结合，从而释放出 Gal(GlcNAc)2 和 pNP 产物。因此，该研究描述了一种新的比色法，可根据从底物中释放出的 pNP 的测定结果对溶菌酶进行定量。

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来源期刊

Accounts of Chemical Research 化学-化学综合

CiteScore

31.40

自引率

1.10%

发文量

312

审稿时长

2 months

期刊介绍： Accounts of Chemical Research presents short, concise and critical articles offering easy-to-read overviews of basic research and applications in all areas of chemistry and biochemistry. These short reviews focus on research from the author’s own laboratory and are designed to teach the reader about a research project. In addition, Accounts of Chemical Research publishes commentaries that give an informed opinion on a current research problem. Special Issues online are devoted to a single topic of unusual activity and significance. Accounts of Chemical Research replaces the traditional article abstract with an article "Conspectus." These entries synopsize the research affording the reader a closer look at the content and significance of an article. Through this provision of a more detailed description of the article contents, the Conspectus enhances the article's discoverability by search engines and the exposure for the research.