Fluorescent substrates for haloalkane dehalogenases: Novel probes for mechanistic studies and protein labeling

化学 费斯特共振能量转移 荧光 紧身衣 活动站点 酶催化 酶动力学 发色团 蛋白质工程 生物物理学 立体化学 组合化学 生物化学 光化学 生物 物理 量子力学
作者
Veronika Brumovska,Esther M. Sánchez‐Carnerero,Zuzana Dunajova,Eduardo Palao,Michaela Slanska,Tomáš Buryška,Jiřı́ Damborský,Petr Klán,Zbyněk Prokop
出处
期刊:Computational and structural biotechnology journal [Elsevier]
卷期号:18: 922-932 被引量:9
标识
DOI:10.1016/j.csbj.2020.03.029
摘要

Haloalkane dehalogenases are enzymes that catalyze the cleavage of carbon-halogen bonds in halogenated compounds. They serve as model enzymes for studying structure–function relationships of >100.000 members of the α/β-hydrolase superfamily. Detailed kinetic analysis of their reaction is crucial for understanding the reaction mechanism and developing novel concepts in protein engineering. Fluorescent substrates, which change their fluorescence properties during a catalytic cycle, may serve as attractive molecular probes for studying the mechanism of enzyme catalysis. In this work, we present the development of the first fluorescent substrates for this enzyme family based on coumarin and BODIPY chromophores. Steady-state and pre-steady-state kinetics with two of the most active haloalkane dehalogenases, DmmA and LinB, revealed that both fluorescent substrates provided specificity constant two orders of magnitude higher (0.14–12.6 μM−1 s−1) than previously reported representative substrates for the haloalkane dehalogenase family (0.00005–0.014 μM−1 s−1). Stopped-flow fluorescence/FRET analysis enabled for the first time monitoring of all individual reaction steps within a single experiment: (i) substrate binding, (ii–iii) two subsequent chemical steps and (iv) product release. The newly introduced fluorescent molecules are potent probes for fast steady-state kinetic profiling. In combination with rapid mixing techniques, they provide highly valuable information about individual kinetic steps and mechanism of haloalkane dehalogenases. Additionally, these molecules offer high specificity and efficiency for protein labeling and can serve as probes for studying protein hydration and dynamics as well as potential markers for cell imaging.
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