Enzymes are widely used in a large number of diverse applications, from laundry detergents to biosensors. As apparent in nature, enzymes associated with surfaces have additional abilities that soluble enzymes lack. Organized, surface-associated enzyme systems can catalyze whole reaction pathways much more efficiently than the same enzymes in solution. In order to incorporate surface-immobilized enzyme systems into useful applications there are challenges that need to be overcome. One is to improve the activities and stabilities of surface-bound enzymes by optimizing their orientations and conformations as well as their surface attachment distances. The other is to control the spatial relationships between the components of multi-enzyme cascades on surfaces aiming to maximize their catalytic efficiencies.
Principal Investigators Neal Woodbury and Hao Yan of Arizona State University believe they know how to accomplish this, and then offer a method to improve the opportunity. They intend to explore peptide space (from known libraries) to identify ligands that will bind to target enzymes and optimize their orientations on surfaces to achieve active functions. This will allow them to create and analyze single- and multi-enzyme systems on peptide-modified solid surfaces. The improvements come by next creating self-assembled DNA nanoscaffold surfaces, which will allow precise control over parameters including inter-enzyme orientation and spacing as well as the distances from enzymes to surfaces or scaffolds. After demonstrating this with the pair of enzymes, a third enzyme can be added for further enhancement.
The combination of peptide anchors/modulators with self-assembled DNA nanostructures represents a unique protein immobilization technology that could significantly improve the activities and stabilities of surface-immobilized enzyme pathways. This will lead to critical advances in a variety of sensing and biocatalytic applications based on multi-step enzyme systems. A major aspect of this proposal will be the kinetic analysis and modeling of enzyme systems assembled on such rationally designed surfaces. The outcome of this proposal will be of particular interest for understanding and designing multi-enzyme reaction pathways in which the ability of one enzyme to directly pass a product to the next is critically dependent on the relative positions of the enzymes involved.
From the broader context, there is an extensive set of potential applications for engineered, self-assembled enzyme systems. In renewable energy, carbon dioxide fixation and bio-diagnostic applications, enzyme-mediated reactions on surfaces will play a significant role. Self-assembled, complex reaction pathways will be of substantial interest in the production of many high-value chemicals, including therapeutics. Thus, improvements of enzyme functions on surfaces would be an important goal to achieve.
This research provides opportunities for student training and outreach to graduates,undergraduates, high school students and teachers. The PIs plan to participate in the Summer High School Internship Program at the Biodesign Institute at ASU where students are exposed to a highly interdisciplinary research environment. The interdisciplinary training opportunity made possible by this project will encourage a spectrum of creative thinking and inspire a greater interest in science and technology.
One of the most exciting technologies to emerge in recent times is programmable molecular assembly: the ability to design a molecular system in a computer and then build a set of components that self-assemble with the programmed shape and function. The most advanced work in this field has been done using DNA, the molecule that makes up our genes, as the programmable molecular material. This emerging technology has been used here to direct the assembly of new catalysts and complexes of catalysts. One example of this that we have generated is a DNA nanostructure with two enzymes attached to it. The first enzyme generates a product which is used by the second enzyme. When these two enzymes are precisely placed such that shells was water molecules that surround them touch, their ability to work together is greatly enhanced; their combined activity increases by more than an order of magnitude beyond what it is when they are allowed to move farther apart. This is not only of great potential use, but also tells us something about how the enzyme complexes in Nature likely work. Nature frequently builds metabolic pathways by grouping enzymes in a pathway together in complexes. In this way, very complicated, multistep synthetic processes can be accomplished with much higher yield than one could do simply in a test tube. This demonstrates the power of nanotechnology, in which control is exerted directly at the level of the molecule, rather than simply at the level of the overall environment.
