Controlling Local Substrate Concentrations in Multi-Enzyme Complexes

In nature we find many examples of bifunctional enzymes that effectively process cascade reactions. For example, the bifunctional enzyme thymidylate synthase-dihydrofolate reductase (TS-DHFR) couples two active sites on the same polypeptide structure via an electrostatic patch. Interactions between the negatively charged cascade intermediate, dihydrofolate, and the positively charged enzyme surface create an environment for bounded diffusion between active sites, thus promoting substrate channeling. This is a useful example from which we can draw design inspiration for synthetic enzyme cascades for multi-step reactions in biofuel cells. To this end, our research group is designing new nanostructured multi-enzyme complexes to study the effects of cascade structure on reaction kinetics. Our overall goal is to turn our understanding of these relationships into a generalized set of design rules that can be used to engineer optimized cascade catalysis. The first step is to investigate interactions between multi-enzyme scaffolds and cascade substrates. The TS-DHFR example suggests that substrate-scaffold interactions are important and can be beneficial to cascade catalysis. Here, we demonstrate that DNA scaffolds can enhance the kinetics of assembled enzymes and that these enhancements are related to the binding energy of the substrate and DNA scaffold. A model system of horseradish peroxidase (HRP) with phenolic substrates and a triangular DNA scaffold (sides ~ 25 nm in length) showed increased enzyme activity with one, two, and three HRPs assembled on the scaffold over freely diffusing HRP modified with short single stranded DNA. Interestingly, the enhancements in activity mimicked the Sabatier principle and a plot of the kinetic enhancement as a function of substrate-DNA binding energy followed the trends of a volcano plot commonly described in heterogeneous catalysis. No enhancement in enzyme activity was observed when the binding of the substrate to the scaffold was weak (k_d = 10 mM). No enhancement occurred when binding was strong (k_d = 4 mM). With intermediate binding (k_d = 100 – 1000 mM) the enhancement in enzyme activity of HRP assembled on a DNA scaffold over freely diffusing enzymes was significant. We hypothesize that the enhancement was due to an increase in local concentration of the substrate resulting from substrate-DNA interactions. We confirm this hypothesis by demonstrating control over the apparent Michaelis constant of HRP-DNA nanostructures by tuning the interactions between substrates and DNA scaffold. These findings represent an important first step in designing multi-enzyme complexes and demonstrate that interactions between substrates and the scaffolds must be considered when engineering such structures. Our current work is focused on extending these findings to controlling the local concentration of enzyme co-factors used in biofuel cell anodes for multi-step oxidation cascades.