(316c) Spatiotemporal Dynamics from a Classic Enzyme Cascade with Self-Governing Substrate Competition

The construction of enzymatic reaction networks in vitro with programmable and predictable dynamics is a central goal of synthetic and systems biology. Biomacromolecules such as genes and enzymes (proteins) are the basic building blocks in the fabrication of such dynamic systems. Despite the abundance of complex dynamics realized with genetic circuits, and even though enzymes play vital roles in the regulation of metabolic pathways and signal transduction processes, the demonstration of enzyme-based networks in vitro has only been explored very recently.^1,2

In this presentation we will introduce a two-enzyme reaction network consisting of a FAD-dependent oxidoreductase and a peroxidase that can generate tunable complex dynamics.³ In a classic enzyme cascade of glucose oxidase (GOx) and horseradish peroxidase (HRP), we discovered a new reaction that GOx can take ABTS^+• as a competitive substrate with oxygen to oxidize glucose. This reaction becomes dominated when the dissolved oxygen is depleting. Based on this, we can generate multiple pulsed responses including the typical charging/discharging, rectangular and parabolic waveforms in a closed system. The waveforms can be well tuned by changing the concentrations of substrates and enzymes. We also demonstrated a safer version of the green bottle experiment with this network, in which the system toggles between colorless and green when the solution is shaken.

In an open system, this reaction network can spontaneously form and display reaction-driven Rayleigh-Bénard convective patterns. The convection is initiated by the accumulating hydrodynamic instability at the air-solution interface where the enzymatic reactions create a denser layer of product. The patterns manifest themselves in green resulting from the interfacial formation and convective flow of ABTS^+•. The spatiotemporal patterns are highly dependent on the depth of the solution and will become increasingly unstable with greater depth.

In summary, we demonstrated the smallest enzymatic reaction network with tunable output waveforms and spatiotemporal pattern formation. The complex dynamic behaviors of this network are based on self-governing substrate competition, rather than activation or inhibition. In addition, the ease of direct observation of convective patterns may also encourage further research on reaction-driven Rayleigh-Bénard convection.

References:

1. Semenov, S. N. et al. Rational design of functional and tunable oscillating enzymatic networks. Nature Chemistry 2015, 7, 160–165.
2. Nijemeisland, M., Abdelmohsen, L. K. E. A., Huck, W. T. S., Wilson, D. A. & van Hest, J. C. M. A compartmentalized out-of-equilibrium enzymatic reaction network for sustained autonomous movement. ACS Central Science 2016, 2, 843–849.
3. Zhang, Y., Tsitkov, S. & Hess, H. Complex dynamics in a two-enzyme reaction network with substrate competition. Nature Catalysis 2018, 1, 276–281.