Interenzyme Substrate Diffusion for an Enzyme Cascade Organized on Spatially Addressable DNA Nanostructures

TLDR: The results suggest that Brownian diffusion of intermediates in solution governed the variations in activity for more distant enzyme pairs, while dimensionally limited diffusion across connected protein surfaces contributed to the enhancement inActivity for closely spaced GOx/HRP assemblies.

Abstract: Spatially addressable DNA nanostructures facilitate the self-assembly of heterogeneous elements with precisely controlled patterns. Here we organized discrete glucose oxidase (GOx)/horseradish peroxidase (HRP) enzyme pairs on specific DNA origami tiles with controlled interenzyme spacing and position. The distance between enzymes was systematically varied from 10 to 65 nm, and the corresponding activities were evaluated. The study revealed two different distance-dependent kinetic processes associated with the assembled enzyme pairs. Strongly enhanced activity was observed for those assemblies in which the enzymes were closely spaced, while the activity dropped dramatically for enzymes as little as 20 nm apart. Increasing the spacing further resulted in a much weaker distance dependence. Combined with diffusion modeling, the results suggest that Brownian diffusion of intermediates in solution governed the variations in activity for more distant enzyme pairs, while dimensionally limited diffusion of interme...