Design and Analysis of Enhanced Catalysis in Scaffolded Multienzyme Cascade Reactions

New developments in nucleic acid nanotechnology and protein scaffold designs have enabled unparalleled control over the spatial organization of synthetic multienzyme cascade reactions. One of the goals of these new technologies is to create nanostructured enzyme cascade reactions that promote substrate channeling along the cascade and, in doing so, enhance cascade catalysis. The concept of substrate channeling has a long and rich history in biochemistry and has established methods of evaluation and quantification. In this Perspective, we review the most common of these methods and discuss them in the context of engineered multienzyme systems and natural bifunctional enzymes with known mechanisms of substrate channeling. In addition, we use experimental data and the results of simulations of coupled-enzyme reactions to develop a set of preliminary design rules for engineering multienzyme nanostructures. The design rules address the limitations on interenzyme distance and active site orientation in substrate channeling and suggest designs for promoting enhanced catalysis, specifically, that enzyme orientation should minimize interenzyme distance and that at distances greater than 1 nm between active sites, significant channeling occurs only if diffusion of the intermediate is bounded through interactions with the surface or scaffold between active sites. This field is rapidly developing and promises to create many more new and exciting technologies.