(12d) Towards Understanding the Kinetic Enhancements in Spatially Organized Multi-Enzyme Structures

In nature, multi-enzyme pathways are often spatially organized into well-defined nanostructures with precise inter-enzyme distances and quaternary structures. Such organization creates the chemical and physical conditions that allows for complex reactions to occur with high specificity and yield in highly heterogeneous environments. Here, we use DNA nanostructures as scaffolds with controlled inter-enzyme distance and shape to study kinetic enhancements in multi-enzyme complexes. The commonly used enzyme horseradish peroxidase (HRP) is used to demonstrate the beneficial effects that DNA scaffolds can impart on single reactions. Additionally, DNA nanostructures and chemical crosslinkers are used to link enzymes in cascade reactions. The glucose oxidase (GOx) and HRP enzyme pair are used to demonstrate the kinetic benefit of spatially organized cascade reactions. The inter-enzyme distances and shapes of multi-enzyme/DNA nanostructures are characterized by Förester resonance energy transfer (FRET) and atomic force microscopy (AFM), respectively. Kinetic enhancements are evaluated by determining the apparent Michaelis-Menton binding constants of the enzyme/DNA nanostructures and by determining the fold enhancement of the initial reaction rates in single and cascade reactions, respectively. In this work we begin to develop a fundamental understanding of the underlining principles that govern the kinetic benefits of spatial organization that are essential to the engineering of multi-enzyme reaction systems.