Role of Dimension and Spatial Arrangement on the Activity of Biocatalytic Cascade Reactions on Scaffolds

Despite broad interest in engineering enzyme cascades on surfaces (i.e., for multistep biocatalysis, enzyme mediated electrocatalysis, biosensing, and synthetic biology), there is a fundamental gap in understanding how the local density and spatial arrangement of enzymes affect overall activity. In this work, the dependence of the overall activity of a cascade reaction on the geometric arrangement and density of enzymes immobilized on a two-dimensional scaffold was elucidated using kinetic Monte Carlo simulations. Simulations were specifically used to track the molecular trajectories of the reaction species and to investigate the turnover frequency of individual enzymes on the surface under diffusion-limited and reaction-limited conditions for random, linear striped, and hexagonal arrangements of the enzymes. Interestingly, the simulation results showed that, under diffusion-limited conditions, the overall cascade activity was only weakly dependent on spatial arrangement and, specifically, nearest-neighbor distance for high enzyme surface coverages. This dependence becomes negligible for reaction-limited conditions, implying that the spatial arrangement has only a minimal impact on cascade activity for the length scales studied here, which has important practical implications. These results suggest that, at short length scales (i.e., sub 10 nm dimensions) and high enzyme densities, sophisticated approaches for controlling enzyme spatial arrangement have little benefit over random immobilization. Moreover, our findings suggest that engineering artificial cascades with enhanced activity will likely require direct molecular channeling rather than a reliance on free molecular diffusion.