Abstract
Maintaining activity of enzymes tethered to solid interfaces remains a major challenge in developing hybrid organic-inorganic devices. In nature, mammalian spermatozoa have overcome this design challenge by having glycolytic enzymes with specialized targeting domains that enable them to function while tethered to a cytoskeletal element. As a step toward designing a hybrid organic-inorganic ATP-generating system, we implemented a biomimetic site-specific immobilization strategy to tether two glycolytic enzymes representing different functional enzyme families: triose phosphoisomerase (TPI; an isomerase) and glyceraldehyde 3-phosphate dehydrogenase (GAPDHS; an oxidoreductase). We then evaluated the activities of these enzymes in comparison to when they were tethered via classical carboxyl-amine crosslinking. Both enzymes show similar surface binding regardless of immobilization method. Remarkably, specific activities for both enzymes were significantly higher when tethered using the biomimetic, site-specific immobilization approach. Using this biomimetic approach, we tethered both enzymes to a single surface and demonstrated their function in series in both forward and reverse directions. Again, the activities in series were significantly higher in both directions when the enzymes were coupled using this biomimetic approach versus carboxyl-amine binding. Our results suggest that biomimetic, site-specific immobilization can provide important functional advantages over chemically specific, but non-oriented attachment, an important strategic insight given the growing interest in recapitulating entire biological pathways on hybrid organic-inorganic devices.
Highlights
A fundamental challenge in developing micro- and nanoscale hybrid material systems is determining how to interface biological components such as enzymes with inorganic surfaces without compromising enzymatic function
In terms of hybrid device engineering, glycolysis has the comparative advantages of needing neither two membrane layers nor a proton-motive force in comparison to mitochondrial oxidative energy production [24]. Because of this comparative simplicity, we hypothesized that a biomimetic approach based on immobilized glycolytic enzymes from sperm would be advantageous in pursuing a long-term objective of designing an ATP-producing system
In conjunction with previous data [16], we have shown a comparative advantage for biomimetic attachment for three broad functional classes of enzymes represented by hexokinase, glucose 6phosphate isomerase (GPI) and triose phosphoisomerase (TPI), and glyceraldehyde 3-phosphate dehydrogenase (GAPDHS)
Summary
A fundamental challenge in developing micro- and nanoscale hybrid material systems is determining how to interface biological components such as enzymes with inorganic surfaces without compromising enzymatic function. Immobilization of enzymes can cause loss of function due to poor accessibility of substrate and/or limited ability to undergo needed conformational changes[1,2,3] To address these difficulties in the context of working toward a bioenergy-producing platform technology, we are employing biomimicry, copying the design of the flagellum of mammalian sperm. These cells have evolved an elegant, high-throughput system for the local production of energy in the form of ATP in the flagellar principal piece. For the enzymes in which targeting domains have been identified, we hypothesize that the strategy of replacing or modifying these domains with a binding or affinity tag would allow tethering of these enzymes in a way that would maximize their function versus standard chemical approaches to binding
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