New Assay Method Based on Raman Spectroscopy for Enzymes Reacting with Gaseous Substrates
New Assay Method Based on Raman Spectroscopy for Enzymes Reacting with Gaseous Substrates
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基于拉曼光谱的酶与气态底物反应的新测定方法
DOI：
10.1002/pro.3569
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发表时间：
2019
期刊：
影响因子：
8
通讯作者：
and Y. Higuchi
中科院分区：
文献类型：
--
作者：
Y. Kawahara‐Nakagawa;K. Nishikawa;S. Nakashima;S. Inoue;T. Ohta;T. Ogura;Y. Shigeta;K. Fukutani;T. Yagi;and Y. Higuchi
通常通过在限定的时间段内定量测量底物和/或产物的初始和最终浓度来测定酶活性。对于涉及气态底物的酶促反应，底物浓度可以直接通过气相色谱法或质谱法估计，或者如果催化反应涉及电子传递与电子介体，表现出氧化还原依赖性光谱变化，则可以间接通过吸收光谱法估计。我们已经开发了一种新的测定系统，用于测量涉及气体底物的酶促反应的时间过程的基础上拉曼光谱。该系统允许以非侵入性方式长时间连续监测反应杯中的气体成分。我们将此系统应用于脱硫弧菌[NiFe]氢化酶的动力学研究。该酶在生理上催化H2的可逆氧化，也具有H/D交换和核自旋异构体转化反应的非生理功能。所提出的系统具有使我们能够同时测量所有氢化酶介导的反应的额外优点。使用所提出的系统，我们证实，H2（完全交换的产品）伴随着HD的H/D交换反应中的D2/H2O系统。基于动力学模型，估算活性位点H/D交换反应速率常数（k）与产物释放速率（kout）之比为1.9 ± 0.2。基于拉曼光谱的分析方法可以应用于其他涉及气体底物的酶的研究。
Enzyme activity is typically assayed by quantitatively measuring the initial and final concentrations of the substrates and/or products over a defined time period. For enzymatic reactions involving gaseous substrates, the substrate concentrations can be estimated either directly by gas chromatography or mass spectrometry, or indirectly by absorption spectroscopy, if the catalytic reactions involve electron transfer with electron mediators that exhibit redox‐dependent spectral changes. We have developed a new assay system for measuring the time course of enzymatic reactions involving gaseous substrates based on Raman spectroscopy. This system permits continuous monitoring of the gas composition in the reaction cuvette in a non‐invasive manner over a prolonged time period. We have applied this system to the kinetic study of the [NiFe] hydrogenase fromDesulfovibrio vulgarisMiyazaki F. This enzyme physiologically catalyzes the reversible oxidation of H2and also possesses the nonphysiological functions of H/D exchange and nuclear spin isomer conversion reactions. The proposed system has the additional advantage of enabling us to measure all of the hydrogenase‐mediated reactions simultaneously. Using the proposed system, we confirmed that H2(the fully exchanged product) is concomitantly produced alongside HD by the H/D exchange reaction in the D2/H2O system. Based on a kinetic model, the ratio of the rate constants of the H/D exchange reaction (k) at the active site and product release rate (kout) was estimated to be 1.9 ± 0.2. The proposed assay method based on Raman spectroscopy can be applied to the investigation of other enzymes involving gaseous substrates.