Difference between revisions of "Team:Aalto-Helsinki/Kinetics"
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<li><a href="#" data-scroll="ter"><h4>Ter</h4></a></li> | <li><a href="#" data-scroll="ter"><h4>Ter</h4></a></li> | ||
<li><a href="#" data-scroll="ycia"><h4>YciA</h4></a></li> | <li><a href="#" data-scroll="ycia"><h4>YciA</h4></a></li> | ||
− | <li><a href="#" data-scroll="car"><h4> | + | <li><a href="#" data-scroll="car"><h4>CAR</h4></a></li> |
<li><a href="#" data-scroll="sfp"><h4>Sfp</h4></a></li> | <li><a href="#" data-scroll="sfp"><h4>Sfp</h4></a></li> | ||
− | <li><a href="#" data-scroll="ado"><h4> | + | <li><a href="#" data-scroll="ado"><h4>ADO</h4></a></li> |
<li><a href="#" data-scroll="other"><h4>Other<br/>constants</h4></a></li> | <li><a href="#" data-scroll="other"><h4>Other<br/>constants</h4></a></li> | ||
<li><a href="#" data-scroll="sources"><h4>Sources</h4></a></li> | <li><a href="#" data-scroll="sources"><h4>Sources</h4></a></li> | ||
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<p>2\(\cdot\)Acetyl-CoA \(\rightarrow\) Acetoacetyl-CoA + CoA</p> | <p>2\(\cdot\)Acetyl-CoA \(\rightarrow\) Acetoacetyl-CoA + CoA</p> | ||
− | <p>AtoB is native to <span style="font-style:italic">Escherichia Coli</span>. The reaction shown above is reversible, but since the ratio of forward and reversible reaction favores strongly the forward one (Vf/Vr: 22.3, Source: [2]) we can approximate is as irreversible.</p> | + | <p>AtoB (acetyl-CoA C-acetyltransferase) is native to <span style="font-style:italic">Escherichia Coli</span>. The reaction shown above is reversible, but since the ratio of forward and reversible reaction favores strongly the forward one (Vf/Vr: 22.3, Source: [2]) we can approximate is as irreversible.</p> |
<p>Based on <a href="http://www.sciencedirect.com/science/article/pii/S0022283605000409">this</a> article, we know that the reaction follows Ping Pong Bi Bi -model and so we get the following rate equation:</p> | <p>Based on <a href="http://www.sciencedirect.com/science/article/pii/S0022283605000409">this</a> article, we know that the reaction follows Ping Pong Bi Bi -model and so we get the following rate equation:</p> | ||
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<p>Acetoacetyl-CoA + NADPH + H\(^+\) \(\rightarrow\) 3-Hydroxybutyryl-CoA + NADP\(^+\)</p> | <p>Acetoacetyl-CoA + NADPH + H\(^+\) \(\rightarrow\) 3-Hydroxybutyryl-CoA + NADP\(^+\)</p> | ||
− | <p>FadB2 is found from<span style="font-style:italic"> Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)</span>. The reaction it catalyzes is reversible and we have assumed it to follow random bi bi reaction model.</p> | + | <p>FadB2 (3-hydroxybutyryl-CoA dehydrogenase) is found from<span style="font-style:italic"> Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)</span>. The reaction it catalyzes is reversible and we have assumed it to follow random bi bi reaction model.</p> |
<p>The equilibrium constant \(K_{eq}\) in reversible random bi bi model is from Haldane relationship \[ K_{eq} = \frac{V_1\cdot K_{M}^{FadB2:3\text{-}Hydroxybutyryl\text{-}CoA}\cdot K_{M}^{FadB2:NADP^+}}{V_2\cdot K_{M}^{FadB2:Acetoacetyl\text{-}CoA}\cdot K_{M}^{FadB2:NADPH}}.\] See [1] for reference. We have not taken H\(^+\) concentration into account in this calculation which is justified because it needs to be fairly constant in the cell or otherwise the cell will die off. This yields us the following as our reaction rate equation.</p> | <p>The equilibrium constant \(K_{eq}\) in reversible random bi bi model is from Haldane relationship \[ K_{eq} = \frac{V_1\cdot K_{M}^{FadB2:3\text{-}Hydroxybutyryl\text{-}CoA}\cdot K_{M}^{FadB2:NADP^+}}{V_2\cdot K_{M}^{FadB2:Acetoacetyl\text{-}CoA}\cdot K_{M}^{FadB2:NADPH}}.\] See [1] for reference. We have not taken H\(^+\) concentration into account in this calculation which is justified because it needs to be fairly constant in the cell or otherwise the cell will die off. This yields us the following as our reaction rate equation.</p> | ||
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<p>Acetoacetyl-CoA + NADPH + H\(^+\) \(\rightarrow\) 3-Hydroxybutyryl-CoA + NADP\(^+\)</p> | <p>Acetoacetyl-CoA + NADPH + H\(^+\) \(\rightarrow\) 3-Hydroxybutyryl-CoA + NADP\(^+\)</p> | ||
− | <p>The enzyme used in the propane pathway is from <span style="font-style:italic">Clostridium acetobutylicum</span>, but only values to be found were for <span style="font-style:italic">Clostridium Kluyveri</span>. However, since the species are very close relatives, we can assume the values to be close enough for comparison.</p> | + | <p>The enzyme used in the propane pathway, Hbd (3-hydroxybutyryl-CoA dehydrogenase), is from <span style="font-style:italic">Clostridium acetobutylicum</span>, but only values to be found were for <span style="font-style:italic">Clostridium Kluyveri</span>. However, since the species are very close relatives, we can assume the values to be close enough for comparison.</p> |
<p>The reaction is reversible, but according to [5], the specific activity of 3-hydroxybutyryl-CoA dehydrogenase (forward) as measured in the direction of acetoacetyl-CoA reduction is 478.6 U/mg protein. The rate of the oxidation reaction (reverse) proceeded with 36.6 U / mg protein. Because of the disparity between these rates we approximate the reaction as irreversible.</p> | <p>The reaction is reversible, but according to [5], the specific activity of 3-hydroxybutyryl-CoA dehydrogenase (forward) as measured in the direction of acetoacetyl-CoA reduction is 478.6 U/mg protein. The rate of the oxidation reaction (reverse) proceeded with 36.6 U / mg protein. Because of the disparity between these rates we approximate the reaction as irreversible.</p> | ||
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<p>3-hydroxybutyryl-CoA \(\rightarrow\) Crotonyl-CoA + H\( _2\)O</p> | <p>3-hydroxybutyryl-CoA \(\rightarrow\) Crotonyl-CoA + H\( _2\)O</p> | ||
− | <p>Crt is found from <span style="font-style:italic;">Clostridium acetobutylicum</span>. Since there is only one substrate in the reaction, we can form the rate equation from basic Michaelis-Menten kinetic model. We assumed the reaction to be irreversible since the enzyme is quite efficient.</p> | + | <p>Crt (3-hydroxybutyryl-CoA dehydratase) is found from <span style="font-style:italic;">Clostridium acetobutylicum</span>. Since there is only one substrate in the reaction, we can form the rate equation from basic Michaelis-Menten kinetic model. We assumed the reaction to be irreversible since the enzyme is quite efficient.</p> |
<p>\[ \frac{K_{cat}^{Crt}\cdot [Crt]\cdot [3\text{-}hydroxybutyryl\text{-}CoA]}{K_{M}^{Crt:3\text{-}Hydroxybutyryl\text{-}CoA} +[3\text{-}hydroxybutyryl\text{-}CoA]} \]</p> | <p>\[ \frac{K_{cat}^{Crt}\cdot [Crt]\cdot [3\text{-}hydroxybutyryl\text{-}CoA]}{K_{M}^{Crt:3\text{-}Hydroxybutyryl\text{-}CoA} +[3\text{-}hydroxybutyryl\text{-}CoA]} \]</p> | ||
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<p>Crotonyl-CoA + NADH + H\( ^+\) \(\rightarrow\) Butyryl-CoA + NAD\( ^+\)</p> | <p>Crotonyl-CoA + NADH + H\( ^+\) \(\rightarrow\) Butyryl-CoA + NAD\( ^+\)</p> | ||
− | <p>Ter is from <span style="font-style:italic;">Treponema denticola</span>. Its reaction without H\( ^+\) is an ordered bi-bi reaction mechanism with NADH binding first [7]. Since we found no references for the reaction to be reversible, we modeled it as irreversible.</p> | + | <p>Ter (trans-2-enoyl-CoA reductase) is from <span style="font-style:italic;">Treponema denticola</span>. Its reaction without H\( ^+\) is an ordered bi-bi reaction mechanism with NADH binding first [7]. Since we found no references for the reaction to be reversible, we modeled it as irreversible.</p> |
<p>\[ \frac{K_{cat}^{Ter}\cdot [Ter] \cdot [Crotonyl\text{-}CoA]\cdot [NADH]}{[Crotonyl\text{-}CoA]\cdot [NADH] + K_{M}^{Ter:NADH}\cdot [Crotonyl\text{-}CoA]+K_{M}^{Ter:Crotonyl\text{-}CoA}\cdot [NADH] + K_{I}^{Ter:Butyryl\text{-}CoA}\cdot K_{M}^{Ter:NADH}} \]</p> | <p>\[ \frac{K_{cat}^{Ter}\cdot [Ter] \cdot [Crotonyl\text{-}CoA]\cdot [NADH]}{[Crotonyl\text{-}CoA]\cdot [NADH] + K_{M}^{Ter:NADH}\cdot [Crotonyl\text{-}CoA]+K_{M}^{Ter:Crotonyl\text{-}CoA}\cdot [NADH] + K_{I}^{Ter:Butyryl\text{-}CoA}\cdot K_{M}^{Ter:NADH}} \]</p> | ||
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<p>Butyryl-CoA + H\( _2\)O \(\rightarrow\) Butyrate + CoA</p> | <p>Butyryl-CoA + H\( _2\)O \(\rightarrow\) Butyrate + CoA</p> | ||
− | <p>YciA is found in <span style="font-style:italic">Haemophilus influenzae</span>. When searching for information about this enzyme no references for it being reversible were found. Because of this we modeled it as irreversible. We know that there is abundance of water in the cell, so when considering rate equation we can safely assume that it doesn't have much effect to it. This is why we can again use the basic Michaelis-Menten rate equation.</p> | + | <p>YciA (acyl-CoA thioester hydrolase) is found in <span style="font-style:italic">Haemophilus influenzae</span>. When searching for information about this enzyme no references for it being reversible were found. Because of this we modeled it as irreversible. We know that there is abundance of water in the cell, so when considering rate equation we can safely assume that it doesn't have much effect to it. This is why we can again use the basic Michaelis-Menten rate equation.</p> |
<p>\[ \frac{K_{cat}^{YciA}\cdot [YciA]\cdot [Butyryl\text{-}CoA]}{K_{M}^{YciA:Butyryl\text{-}CoA} +[Butyryl\text{-}CoA]} \]</p> | <p>\[ \frac{K_{cat}^{YciA}\cdot [YciA]\cdot [Butyryl\text{-}CoA]}{K_{M}^{YciA:Butyryl\text{-}CoA} +[Butyryl\text{-}CoA]} \]</p> | ||
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− | <!-- | + | <!-- CAR --> |
<section id="car" data-anchor="car"> | <section id="car" data-anchor="car"> | ||
− | <h2> | + | <h2>CAR</h2> |
<p>Butyrate + NADPH + ATP \(\rightarrow\) Butyraldehyde + NADP\(^+\) + AMP + 2P\(_i\)</p> | <p>Butyrate + NADPH + ATP \(\rightarrow\) Butyraldehyde + NADP\(^+\) + AMP + 2P\(_i\)</p> | ||
− | <p> | + | <p>CAR-enzyme (carboxylic acid reductase) is originally from <span style="font-style:italic">Mycobacterium marinum</span>. We assumed that this reaction is irreversible, which is justified because we have ATP in the reactants so we know that the possible reverse reaction can’t be very efficient. |
For the same reasons as mentioned before, we didn’t consider \(H^+\) in equations. <a href="http://www.pnas.org/content/110/1/87">We know</a> that the reaction can be modeled using Bi Uni Uni Bi Ping Pong mechanism. Thus, the rate equation will be</p> | For the same reasons as mentioned before, we didn’t consider \(H^+\) in equations. <a href="http://www.pnas.org/content/110/1/87">We know</a> that the reaction can be modeled using Bi Uni Uni Bi Ping Pong mechanism. Thus, the rate equation will be</p> | ||
− | <p>\[\frac{K_{cat}^{ | + | <p>\[\frac{K_{cat}^{CAR}\cdot [CAR]\cdot [Butyrate]\cdot [NADPH]\cdot [ATP]}{K_{M}^{CAR:Butyrate}\cdot K_{M}^{CAR:NADPH}\cdot [ATP]+K_{M}^{CAR:ATP}\cdot [Butyrate]\cdot [NADPH]+K_{M}^{CAR:NADPH}\cdot [Butyrate]\cdot [ATP]}\]\[\cdots \frac{}{+K_{M}^{CAR:Butyrate}\cdot [NADPH]\cdot [ATP]+ [Butyrate]\cdot [NADPH]\cdot [ATP]}\]</p> |
<table class="table table-bordered"> | <table class="table table-bordered"> | ||
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<tbody> | <tbody> | ||
<tr> | <tr> | ||
− | <td><p>\( K_{cat}^{ | + | <td><p>\( K_{cat}^{CAR} \)</p></td> |
<td><p>150 1/min</p></td> | <td><p>150 1/min</p></td> | ||
<td><p>[9]</p></td> | <td><p>[9]</p></td> | ||
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</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td><p>\( K_{M}^{ | + | <td><p>\( K_{M}^{CAR:Butyrate} \)</p></td> |
<td><p>0.013 mol/l</p></td> | <td><p>0.013 mol/l</p></td> | ||
<td><p>[9]</p></td> | <td><p>[9]</p></td> | ||
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</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td><p>\( K_{M}^{ | + | <td><p>\( K_{M}^{CAR:NADPH} \)</p></td> |
<td><p>4.8e-05 mol/l</p></td> | <td><p>4.8e-05 mol/l</p></td> | ||
<td><p>[9]</p></td> | <td><p>[9]</p></td> | ||
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</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td><p>\( K_{M}^{ | + | <td><p>\( K_{M}^{CAR:ATP} \)</p></td> |
<td><p>0.000115 mol/l</p></td> | <td><p>0.000115 mol/l</p></td> | ||
<td><p>[9]</p></td> | <td><p>[9]</p></td> | ||
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</section> | </section> | ||
− | <!-- | + | <!-- CAR ends --> |
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<section id="sfp" data-anchor="sfp"> | <section id="sfp" data-anchor="sfp"> | ||
<h2>Sfp</h2> | <h2>Sfp</h2> | ||
− | <p>Sfp does not directly affect to the intermediates in our pathway, but instead acts as an activating enzyme for | + | <p>Sfp (4'-phosphopantetheinyl transferase) does not directly affect to the intermediates in our pathway, but instead acts as an activating enzyme for CAR. We have modeled the reactions concerning Sfp <a href="https://2015.igem.org/Team:Aalto-Helsinki/Car-activation">here</a>.</p> |
</section> | </section> | ||
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− | <!-- | + | <!-- ADO --> |
<section id="ado" data-anchor="ado"> | <section id="ado" data-anchor="ado"> | ||
− | <h2> | + | <h2>ADO</h2> |
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<p>Since we could not model the reactions that govern ADO's function, we approximated these reactions by simplifying the enzyme kinetics that govern ADO to the simplest case of Miclaelis-Menten kinetics. While this is not ideal, with current data and within these time limitations we can't make better assumptions.</p> | <p>Since we could not model the reactions that govern ADO's function, we approximated these reactions by simplifying the enzyme kinetics that govern ADO to the simplest case of Miclaelis-Menten kinetics. While this is not ideal, with current data and within these time limitations we can't make better assumptions.</p> | ||
− | <p>\[ \frac{K_{cat}^{ | + | <p>\[ \frac{K_{cat}^{ADO}\cdot [ADO]\cdot [Butyrate]}{K_{M}^{ADO:Butyrate} +[Butyrate]} \]</p> |
<table class="table table-bordered"> | <table class="table table-bordered"> | ||
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<tbody> | <tbody> | ||
<tr> | <tr> | ||
− | <td><p>\( K_{cat}^{ | + | <td><p>\( K_{cat}^{ADO} \)</p></td> |
<td><p>0.03 1/min</p></td> | <td><p>0.03 1/min</p></td> | ||
<td><p>[10]</p></td> | <td><p>[10]</p></td> | ||
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</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td><p>\( K_{M}^{ | + | <td><p>\( K_{M}^{ADO:Butyraldehyde} \)</p></td> |
<td><p>0.0101 mol/l</p></td> | <td><p>0.0101 mol/l</p></td> | ||
<td><p>[10]</p></td> | <td><p>[10]</p></td> | ||
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</section> | </section> | ||
− | <!-- | + | <!-- ADO ends --> |
Revision as of 22:41, 18 September 2015