Coupled enzyme reactions performed in heterogeneous reaction media: experiments and modeling for glucose oxidase and horseradish peroxidase in a PEG/citrate aqueous two-phase system.
Coupled enzyme reactions performed in heterogeneous reaction media: experiments and modeling for glucose oxidase and horseradish peroxidase in a PEG/citrate aqueous two-phase system.
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DOI：
10.1021/jp501126v
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发表时间：
2014-03-06
期刊：
影响因子：
--
通讯作者：
Keating CD
中科院分区：
文献类型：
--
作者：
Aumiller WM Jr;Davis BW;Hashemian N;Maranas C;Armaou A;Keating CD
发生生物反应的细胞内环境充满了大分子，并细分为物理性质和化学组成不同的微环境。这里描述的工作结合了实验和计算模型系统，以帮助理解这种异质反应介质对偶联酶反应结果的影响。我们的实验模型系统的解决方案的异质性是一个两相聚乙二醇（PEG）/柠檬酸钠的水溶液混合物，提供共存的PEG丰富和柠檬酸盐丰富的阶段。研究了PEG/柠檬酸盐双水相体系中葡萄糖氧化酶（GOX）与辣根过氧化物酶（HRP）偶联反应的动力学。酶动力学不同的两个阶段，特别是HRP。这两种酶，以及底物葡萄糖和H2 O2，分配到富含柠檬酸盐的阶段，然而，Amplex红底物必须完成的顺序反应强烈分配到富含PEG的阶段。ATPS中的反应通过结合测量的分配和动力学参数的数学模型定量描述。然后将该模型扩展到新的反应条件，即，较高的酶浓度。实验和计算结果表明，跨界面的传质对于维持所观察到的产物形成速率是至关重要的，这可能是体内代谢调节的一种手段。虽然特定系统的结果将取决于酶反应和微环境的细节，但这项工作演示了如何在复杂的非均相介质中耦合酶反应可以用数学模型来理解。
The intracellular environment in which biological reactions occur is crowded with macromolecules and subdivided into microenvironments that differ in both physical properties and chemical composition. The work described here combines experimental and computational model systems to help understand the consequences of this heterogeneous reaction media on the outcome of coupled enzyme reactions. Our experimental model system for solution heterogeneity is a biphasic polyethylene glycol (PEG)/sodium citrate aqueous mixture that provides coexisting PEG-rich and citrate-rich phases. Reaction kinetics for the coupled enzyme reaction between glucose oxidase (GOX) and horseradish peroxidase (HRP) were measured in the PEG/citrate aqueous two-phase system (ATPS). Enzyme kinetics differed between the two phases, particularly for the HRP. Both enzymes, as well as the substrates glucose and H2O2, partitioned to the citrate-rich phase; however, the Amplex Red substrate necessary to complete the sequential reaction partitioned strongly to the PEG-rich phase. Reactions in ATPS were quantitatively described by a mathematical model that incorporated measured partitioning and kinetic parameters. The model was then extended to new reaction conditions, i.e., higher enzyme concentration. Both experimental and computational results suggest mass transfer across the interface is vital to maintain the observed rate of product formation, which may be a means of metabolic regulation in vivo. Although outcomes for a specific system will depend on the particulars of the enzyme reactions and the microenvironments, this work demonstrates how coupled enzymatic reactions in complex, heterogeneous media can be understood in terms of a mathematical model.
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DOI：
10.1016/j.cep.2007.08.006
发表时间：
2008-01-01
影响因子：
4.3
作者：
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通讯作者：
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影响因子：
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作者：
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影响因子：
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作者：
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通讯作者：
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影响因子：
3.4
作者：
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通讯作者：
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影响因子：
4.3
作者：
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