"
Team:Nevada/Modeling
From 2009.igem.org
| Line 106: | Line 106: | ||
</html> | </html> | ||
| - | We developed a computer model for measuring enzyme kinetics of our pathway <html><a href="#FIG1">(Figure 1)</a | + | We developed a computer model for measuring enzyme kinetics of our pathway <html><a href="#FIG1">(Figure 1)</a>. The model was developed using <i> <a href="http://www.wolfram.com/">Mathematica</a></i></html> based upon steady-state Michaelis–Menten kinetics <html><a href="#FIG2">(Figure 2)</a></html>. It revealed that the kinetic parameters for the wild type isoform of 4-coumarate:CoA ligase were creating a bottleneck and significantly slowing down the overall reaction <html><a href="#FIG3">(Figure 3)</a></html>. There are two basic ways to get around this problem: (1) to use a mutant enzyme <html><a href="#FIG4">(Figure 4)</a></html> with more favorable kinetic parameters or to increase the concentration of the wild type isoform of 4-coumarate:CoA ligase <html><a href="#FIG5">(Figure 5)</a></html>. For all of the kinetics characterized assume all of the enzymes are in equal concentrations, except for the model involving 10x the concentration of the wild type isoform of 4-coumarate:CoA ligase <html><a href="#FIG5">(Figure 5)</a></html>. |
We chose to investigate both of these methods to insure no ill-conceived reaction kinetics were occurring. | We chose to investigate both of these methods to insure no ill-conceived reaction kinetics were occurring. | ||
Revision as of 02:45, 22 October 2009
We developed a computer model for measuring enzyme kinetics of our pathway (Figure 1). The model was developed using Mathematica based upon steady-state Michaelis–Menten kinetics (Figure 2). It revealed that the kinetic parameters for the wild type isoform of 4-coumarate:CoA ligase were creating a bottleneck and significantly slowing down the overall reaction (Figure 3). There are two basic ways to get around this problem: (1) to use a mutant enzyme (Figure 4) with more favorable kinetic parameters or to increase the concentration of the wild type isoform of 4-coumarate:CoA ligase (Figure 5). For all of the kinetics characterized assume all of the enzymes are in equal concentrations, except for the model involving 10x the concentration of the wild type isoform of 4-coumarate:CoA ligase (Figure 5). We chose to investigate both of these methods to insure no ill-conceived reaction kinetics were occurring.
While we have a hypothetical model for the reverse reaction, it is purely speculative as no real world data exists characterizing the reverse reaction rates of the aforementioned enzymes. This also means our model does not account for reverse reaction kinetics due to the lack of available data. We also used kinetic values from a highly conserved variant of Cinnamoyl-CoA reductase called Feruloyl-CoA reductase. Data for the latter was obtained from the Arabidopsis thaliana isoform and is structurally similar enough to act as a proper substitute for Cinnamoyl-CoA reductase (Table 1).
Figure 1 - Cinnamaldehyde Synthesis Pathway
Figure 2 - Steady State Model of Cinnamaldehyde Production from an L-Phenylalanine Precursor using the wild type isoform of 4-coumarate:CoA ligase
Figure 3 - Steady State Model of Cinnamaldehyde Production from an L-Phenylalanine Precursor using the mutant isoform of 4-coumarate:CoA ligase
Figure 4 - Steady State Model of Cinnamaldehyde Production from an L-Phenylalanine Precursor using 10 times the concentration of the wild type isoform of 4-coumarate:CoA ligase
Figure 5 - Modeling Source Code
Table 1 - Enzyme Modeling Kinetics and Environmental Conditions
References
1. Mulquiney, P.J., Kuchel, P.W. Modeling Metabolism with Mathematica. CRC Press: 2003.
2. Schneider, K., Hovel, K., Witzel, K., Hamberger, B., Schombur, D., Kombrink, E., Stuible, H.P. 2003. The substrate specificity-determining amino acid code of 4-coumarate:CoA ligase. PNAS. 100, 8601-8606.
3. Stuible, H.P., Buttner, D., Ehlting, J., Hahlbrock, K., Kombrink, E. 2000. Mutational analysis of 4-coumarate:CoA ligase identifies functionally important amino acids and verifies its close relationship to other adenylate-forming enzymes. FEBS Letters. 467, 117-122.
