Assemblase Kinectics
Overview
After achieving one of our molecular dynamic (MD) objectives; to find the distance between the active sites of our Assemblase system, a new distance parameter was used in the Enzyme Kinetics and Diffusion model created by last year's iGEM team. The model primarily analyses the effect of the distance between enzymes has on reaction rate in a two-step biochemical pathway. The enzymes reaction rates follow Michaelis-Menten kinetics whilst the diffusion of the intermediary substrate acts in accordance with Fick's Law. The full explanation of their model including its assumptions and the manner in which it represents two-step enzyme reactions can be found on their wiki here. The model was adjusted to incorporate the kinetic parameters of our Assemblase's enzymes, that were discovered from engaging with the literature1,2. We hypothesised that we would receive similar results as last years team - a confirmation that co-localising our enzymes would drive substrate channeling and increase reaction yield despite our different kinetic parameters.
Results
Over 0.2 seconds the concentration of intermediary substrate reaching the second enzyme in the pathway was measured at multiple distances. The largest distance between the first and second enzymes was 1460 angstroms with this distance being incrementally reduced by 40 angstroms to the smallest distance of 20 angstroms. Subsequently, the model enabled us to see the effect distance has on reaction yield (fig. 1). Certainly, steady-state Paclitaxel production is shown to have been achieved faster when the enzymes are in closer proximity, validating our scaffold design.
Figure 1. From right to left, each coloured line represents a smaller distance between the two enzymes. The uppermost line (orange) shows the concentration-time curve when the enzymes are separated by a distance of 20 angstroms, with the lowermost line displaying the concentration-time curve for a distance of 1460 angstroms.
The model integrates under each distance curve (fig. 1), facilitating a comparison of the amount product formed for each distance, over the same time period (fig.2). A clear non-linear relationship is apparent, with increasingly higher yields of Paclitaxel produced for shorter distances between the two enzymes (fig. 2). The distance estimate from our MD analysis, 150 angstroms, puts the yield of our Assemblase system to 8 to 9 micromoles of Paclitaxel over the 0.2 second time-frame of the model. However, more convincingly we learnt from MD that our enzymes active sites are kept in a range of approximately between 50 to 240 angstroms - ensuring a yield no less than 6 micromoles over 0.2 seconds (fig. 2).
Figure 2. Every point represents the amount of Paclitaxel produced in micromoles when the enzymes are separated by a certain distance in angstroms.
Conclusion
In summary, as expected, the Enzyme Kinetic and Diffusion Model, developed by last year's UNSW iGEM team verified our scaffolding design. It demonstrated that clustering enzymes in close proximity of each other, such as on our Assemblase system, achieves increased yields of Paclitaxel.
References
- H.L. Cheng, R.Y. Zhao, T.J. Chen, W.B. Yu, F. Wang, K.D. Cheng, and P. Zhu, Cloning and Characterization of the Glycoside Hydrolases That Remove Xylosyl Groups from 7-  -xylosyl-10-deacetyltaxol and Its Analogues. Molecular &Cellular Proteomics 12: pp.2236–2248, 2013.
- B.J. Li, H. Wang, T. Gong, J.J. Chen, T.J. Chen, J.L. Yang & P. Zhu, Improving 10-deacetylbaccatin III-10-b-O-acetyltransferase catalytic fitness for Taxol production. Nature Communications 8, 15544 doi: 10.1038/ncomms15544 (2017).