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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
[Image: see text] 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...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Chemical
Society
2014
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3983363/ https://www.ncbi.nlm.nih.gov/pubmed/24517887 http://dx.doi.org/10.1021/jp501126v |
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author | Aumiller, William M. Davis, Bradley W. Hashemian, Negar Maranas, Costas Armaou, Antonios Keating, Christine D. |
author_facet | Aumiller, William M. Davis, Bradley W. Hashemian, Negar Maranas, Costas Armaou, Antonios Keating, Christine D. |
author_sort | Aumiller, William M. |
collection | PubMed |
description | [Image: see text] 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 H(2)O(2), 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. |
format | Online Article Text |
id | pubmed-3983363 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | American Chemical
Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-39833632015-02-11 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 Aumiller, William M. Davis, Bradley W. Hashemian, Negar Maranas, Costas Armaou, Antonios Keating, Christine D. J Phys Chem B [Image: see text] 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 H(2)O(2), 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. American Chemical Society 2014-02-11 2014-03-06 /pmc/articles/PMC3983363/ /pubmed/24517887 http://dx.doi.org/10.1021/jp501126v Text en Copyright © 2014 American Chemical Society |
spellingShingle | Aumiller, William M. Davis, Bradley W. Hashemian, Negar Maranas, Costas Armaou, Antonios Keating, Christine D. 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 |
title | 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 |
title_full | 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 |
title_fullStr | 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 |
title_full_unstemmed | 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 |
title_short | 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 |
title_sort | 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 |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3983363/ https://www.ncbi.nlm.nih.gov/pubmed/24517887 http://dx.doi.org/10.1021/jp501126v |
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