The past decade has seen many reports of enhanced enzyme cascade activity due to nanoscale proximity enabled by DNA nanotechnology. The commonly accepted explanation for the enhancement enzyme proximity, which facilitates channeling, i.e. the rapid transport of intermediate substrate to the next enzyme downstream. Utilizing glucose oxidase (GOx) and horseradish peroxidase (HRP) as a model cascade system, we first studied the kinetics of free GOx-HRP cascade. The diffusion time of the intermediate in the rate of the free enzyme cascade was negligible. Therefore, a permanent activity enhancement requires an increase in the activity of the rate-limiting enzyme. We then directly tested the channeling effect using the GOx-HRP conjugate and found no cascade activity enhancement. Moreover, the production by the conjugate could be completely suppressed by a competing enzyme, catalase. These results indicate that the proximity effect does not enhance the throughput of an enzyme cascade. We hypothesize that the DNA nanostructure drives the enhancement: the structure’s negative charge lowers the local pH thus improving the individual enzyme activity. To prove our hypothesis, we demonstrated increased throughput of a new cascade consisting of D-amino acid oxidase (DAAO) and cytochrome c (Cyt c) by engineering the microenvironment of one enzyme. DAAO and Cyt c are more active in alkaline and acidic environments, respectively. To allow both enzymes to operate optimally under alkaline conditions, we conjugated Cyt c with a negatively charged polyelectrolyte to create a more acidic local microenvironment for the Cyt c. This achieved at least 10-fold enhancement in the overall production. To our knowledge, it is the first demonstration of deliberate control of the pH condition to optimize individual enzyme activity in a cascade. Our findings challenge the concept of proximity channeling in artificial enzyme cascades and provide a novel strategy to overlap the operating pH range for cascade enzymes, which is essential for in vitro one-pot multienzyme catalysis.

Microenvironment effect rather than proximity channeling causes the activity enhancement in enzyme cascade

253rd National Meeting (2017)

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