Modulation of Enzyme Cascade Activity by Local Substrate Enrichment and Exclusion on DNA Nanostructures

Area of Science:

• Biochemistry
• Nanotechnology
• Synthetic Biology

Background:

• Substrate confinement and channeling are crucial for multienzyme pathways.
• These factors influence catalytic efficiency and specificity in nanoreactors.

Purpose of the Study:

• To modulate a multienzyme system's cascade activity through surface affinity binding.
• To investigate the impact of substrate enrichment on enzyme nanoreactors.

Main Methods:

• Utilized DNA origami modified with aptamers to bind and enrich ATP substrates.
• Immobilized enzymes on DNA nanostructures for cascade reactions.
• Analyzed enzyme kinetics using Michaelis-Menten parameters.

Main Results:

• Enhanced enzyme cascade activity by over 2-fold using aptamer-modified DNA nanostructures.
• Observed a ~3-fold decrease in apparent K_M with substrate enrichment, indicating higher effective substrate concentration.
• Blocked aptamers reduced activity and increased apparent K_M, suggesting substrate exclusion.

Conclusions:

• Surface affinity binding and local substrate enrichment significantly enhance multienzyme cascade activity.
• This strategy is applicable to various immobilized multienzyme systems.
• Developed a method for improving biomimetic nanoreactor design and efficiency.