Team:ITESM-CEM/EnzymaticKinectics
From 2014.igem.org
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<h2>Overview</h2> | <h2>Overview</h2> | ||
<p style="text-align: justify; text-justify: inter-word;">During 2014, iGEM ITESM CEM Team worked on the development of a metabolic pathway for 7-ketocholesterol degradation by enzyme therapy of human macrophages, using two hypothetic enzymes of the microorganism Rhodococcus jostii, active when the microbe grows using 7-ketocholesterol as a sole source of carbon. These enzymes (oxoacyl reductase and 7-dehydratase) were first described as potentially being used to catalyse these reactions by Mathieu (1); and were cloned and purified by iGEM ITESM CEM Team using E. coli and a diverse array of expression vectors.<br> | <p style="text-align: justify; text-justify: inter-word;">During 2014, iGEM ITESM CEM Team worked on the development of a metabolic pathway for 7-ketocholesterol degradation by enzyme therapy of human macrophages, using two hypothetic enzymes of the microorganism Rhodococcus jostii, active when the microbe grows using 7-ketocholesterol as a sole source of carbon. These enzymes (oxoacyl reductase and 7-dehydratase) were first described as potentially being used to catalyse these reactions by Mathieu (1); and were cloned and purified by iGEM ITESM CEM Team using E. coli and a diverse array of expression vectors.<br> | ||
- | However, in order to properly asses and predict the behaviour of both proteins in the cytosol of human cells, it is first necessary to numerically model their interaction and catalysis over their substrates. In order to do so, the proposed pathway must firstly be analysed.<br><br> | + | However, in order to properly asses and predict the behaviour of both proteins in the cytosol of human cells, it is first necessary to numerically model their interaction and catalysis over their substrates. In order to do so, the proposed pathway must firstly be analysed.<br><br></p> |
- | <img src="https://static.igem.org/mediawiki/2014/6/6f/Ruta_metab%C3%B3lica_sin_fondo.jpg" align="left" width="600" height="423" hspace="10" BORDER=10><br><br><br><br><br><br><br><br><br><br> <br><br><br><br><br><br><br><br><br><br><br>< | + | <p class="centeredImage"><img src="https://static.igem.org/mediawiki/2014/6/6f/Ruta_metab%C3%B3lica_sin_fondo.jpg" align="left" width="600" height="423" hspace="10" BORDER=10><br><br><br><br><br><br><br><br><br><br> <br><br><br><br><br><br><br><br><br><br><br></p> |
<pie><b>Figure 1.-</b> Theoretical metabolic pathway for 7-ketocholesterol degradation in Rhodococcus jostii. Red arrows indicated the most intuitive order of reactions.</pie><br><br> | <pie><b>Figure 1.-</b> Theoretical metabolic pathway for 7-ketocholesterol degradation in Rhodococcus jostii. Red arrows indicated the most intuitive order of reactions.</pie><br><br> | ||
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The following system of differential equations was generated, where each compound has a particular abbreviation (7KC for 7-ketocholesterol, 7βOHC for 7-βOH-Cholesterol, and Ch for Cholesterol: </p><br> | The following system of differential equations was generated, where each compound has a particular abbreviation (7KC for 7-ketocholesterol, 7βOHC for 7-βOH-Cholesterol, and Ch for Cholesterol: </p><br> | ||
- | <p class="centeredImage"><img src="https://static.igem.org/mediawiki/2014/f/fa/Formula_2_editado-1.jpg" hspace="20" | + | <p class="centeredImage"><img src="https://static.igem.org/mediawiki/2014/f/fa/Formula_2_editado-1.jpg" hspace="20"></p><br> |
<p style="text-align: justify; text-justify: inter-word;"> This means that the concentration of 7-ketocholesterol is always described by the Michaelis-Menten reaction rate of the first enzyme, which tends to degrade it (a zero term stands for the non-existing 7-ketocholesterol synthesis); the concentration of 7- βOH-cholesterol is described by both the first and the second enzyme Micahelis-Menten kinetics, where the first one models its synthesis and the second one its degradation. Finally, cholesterol concentration is zero in the first stage of the reaction (when 7- βOH-cholesterol has not been metabolized yet), and is modelled by the second enzyme kinetics in the second reaction stage; here, it is generated at a reaction rate of the same magnitude and opposite sign of that of 7-βOH-Cholesterol degradation (a zero term stands for the non-existing cholesterol metabolism).<br> | <p style="text-align: justify; text-justify: inter-word;"> This means that the concentration of 7-ketocholesterol is always described by the Michaelis-Menten reaction rate of the first enzyme, which tends to degrade it (a zero term stands for the non-existing 7-ketocholesterol synthesis); the concentration of 7- βOH-cholesterol is described by both the first and the second enzyme Micahelis-Menten kinetics, where the first one models its synthesis and the second one its degradation. Finally, cholesterol concentration is zero in the first stage of the reaction (when 7- βOH-cholesterol has not been metabolized yet), and is modelled by the second enzyme kinetics in the second reaction stage; here, it is generated at a reaction rate of the same magnitude and opposite sign of that of 7-βOH-Cholesterol degradation (a zero term stands for the non-existing cholesterol metabolism).<br> | ||
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<p class="centeredImage"><img src="https://static.igem.org/mediawiki/2014/2/2e/Mathematical_Model-3.jpg" height="187" width="334" hspace="20" BORDER=10></p><br> | <p class="centeredImage"><img src="https://static.igem.org/mediawiki/2014/2/2e/Mathematical_Model-3.jpg" height="187" width="334" hspace="20" BORDER=10></p><br> | ||
- | <p><pie><b>Figure 2.-<b> Plots generated by the numerical solution of the differential model of enzyme kinetics using Runge-Kutta’s fourth order method. Decreasing values for Michaelis constant for the first reaction are shown in each plot.</pie></p> | + | <p><pie><b>Figure 2.-</b> Plots generated by the numerical solution of the differential model of enzyme kinetics using Runge-Kutta’s fourth order method. Decreasing values for Michaelis constant for the first reaction are shown in each plot.</pie></p> |
<p style="text-align: justify; text-justify: inter-word;"> The first plot might accurately represent the situation inside human macrophages, where 7-ketocholesterol cannot be degraded at an appropriate rate, and gradually accumulates. If we further consider that this model does not include the endogenous synthesis and the uptake of 7-ketocholesterol, we can easily see why this molecule’s concentration rapidly builds up in the bloodstream. However, as Km values for this reaction diminish, the plots tend to represent more efficient metabolic pathways: we expect our synthetic route, based of Rhodococcus jostii’s sterol metabolism, to be modelled by one of the last two plots in the figure; here, 7-ketocholesterol is degraded faster, and regular cholesterol, which can readily be metabolized by human enzymes, is produced.</p> | <p style="text-align: justify; text-justify: inter-word;"> The first plot might accurately represent the situation inside human macrophages, where 7-ketocholesterol cannot be degraded at an appropriate rate, and gradually accumulates. If we further consider that this model does not include the endogenous synthesis and the uptake of 7-ketocholesterol, we can easily see why this molecule’s concentration rapidly builds up in the bloodstream. However, as Km values for this reaction diminish, the plots tend to represent more efficient metabolic pathways: we expect our synthetic route, based of Rhodococcus jostii’s sterol metabolism, to be modelled by one of the last two plots in the figure; here, 7-ketocholesterol is degraded faster, and regular cholesterol, which can readily be metabolized by human enzymes, is produced.</p> |
Revision as of 05:54, 14 October 2014
ITESM-CEM | Enzy7-K me |
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