Cascade reactions drive and regulate a variety of metabolic activities. Efficient coupling of substrate transport between enzymes is important for overall pathway activity and also controls the depletion of intermediate molecules that drive the reaction forward. Here, we assembled a three-enzyme pathway on a series of DNA nanoscaffolds to investigate the dependence of their activities on spatial arrangement. Unlike previous studies, the overall activity of the three-enzyme pathway relied less on inter-enzyme distance and more on the geometric patterns that arranged them within a relatively small range of 10-30 nm. Pathway intermediate detection demonstrated that the assembled enzyme systems quickly depleted the intermediate molecules through efficient reaction coupling.
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