Kinetics of enzyme‐catalysed oxygen isotope exchange between phosphate and water revealed by Raman spectroscopy
Kinetics of enzyme‐catalysed oxygen isotope exchange between phosphate and water revealed by Raman spectroscopy
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拉曼光谱揭示酶催化磷酸盐和水之间氧同位素交换的动力学
DOI：
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发表时间：
2017
期刊：
影响因子：
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通讯作者：
E. Frossard
中科院分区：
文献类型：
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作者：
C. Sperber;H. Lewandowski;F. Tamburini;S. Bernasconi;W. Amelung;E. Frossard
焦磷酸酶（EC 3.6.1.1）是催化焦磷酸盐水解的普遍存在的酶，焦磷酸盐是许多生物化学反应的副产物。水解导致新形成的磷酸盐分子和水之间的氧同位素交换。在这里，我们应用拉曼光谱监测的氧同位素交换反应中存在的焦磷酸酶从面包酵母。为此目的，酶测定组成的0.8 m 18 O-富集磷酸盐在pH缓冲条件下制备。加入焦磷酸酶后，溶液的拉曼光谱立即开始向更高的波数移动，表明磷酸盐中的18 O逐渐被水中的16 O取代。通过将Voigt函数拟合到测量的拉曼光谱来量化分析结果，所述拉曼光谱允许确定溶液中每种磷酸盐同位素体随时间的相对贡献。基于不同磷酸盐物质的相对贡献，可以假设一级动力学来计算表观总氧交换速率。不同的磷酸盐同位素体的逐步形成和消失，然后通过应用一个连续的反应方案与一阶步骤建模。实验结果表明，拉曼光谱可用于磷酸盐-水体系中酶催化氧同位素交换动力学的研究。版权所有© 2016约翰威利父子有限公司.
Pyrophosphatases (EC 3.6.1.1) are ubiquitous enzymes that catalyse the hydrolysis of pyrophosphate, a byproduct of many biochemical reactions. The hydrolysis leads to an oxygen isotope exchange between the newly formed phosphate molecules and water. Here, we applied Raman spectroscopy to monitor the oxygen isotope exchange reaction in presence of pyrophosphatase from baker's yeast. For this purpose, enzymatic assays consisting of 0.8 m 18O-enriched phosphate were prepared under pH-buffered conditions. Upon addition of pyrophosphatase, the Raman spectrum of the solution immediately started to shift to higher wavenumbers, indicating the progressive substitution of 18O in phosphate by 16O from water. The analytical results were quantified by fitting a Voigt function to the measured Raman spectra that allowed to determine the relative contribution of each phosphate isotopologue in solution over time. Based on the relative contribution of the different phosphate species, the apparent overall oxygen exchange rate could be calculated assuming a first-order kinetic. The progressive formation and disappearance of the different phosphate isotopologues were then modelled by applying a consecutive reaction scheme with first-order steps. The results of our experiments show that Raman spectroscopy can be used to study the kinetics of enzyme-catalysed oxygen isotope exchange in the phosphate–water system. Copyright © 2016 John Wiley & Sons, Ltd.
影响因子：
56.9
作者：
RADZICKA, A;WOLFENDEN, R
通讯作者：
WOLFENDEN, R