Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, New York, United States of America.
PLoS One. 2013 Apr 23;8(4):e61434. doi: 10.1371/journal.pone.0061434. Print 2013.
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.
将酶固定在固体界面上保持活性仍然是开发混合有机-无机器件的主要挑战。在自然界中，哺乳动物精子通过具有特殊靶向结构域的糖酵解酶克服了这一设计挑战，使它们能够在与细胞骨架元素连接的情况下发挥作用。作为设计混合有机-无机 ATP 生成系统的一步，我们实施了一种仿生定点固定化策略，将两种代表不同功能酶家族的糖酵解酶固定在一起：磷酸丙糖异构酶（TPI；异构酶）和甘油醛 3-磷酸脱氢酶（GAPDHS；氧化还原酶）。然后，我们将这些酶的活性与通过经典羧基-胺交联固定时的活性进行了比较。无论固定方法如何，两种酶的表面结合都相似。值得注意的是，当使用仿生、定点固定化方法固定时，两种酶的比活性都显著提高。使用这种仿生方法，我们将两种酶都固定在单个表面上，并证明了它们在正向和反向两个方向上的串联功能。同样，当使用这种仿生方法偶联时，两种酶在两个方向上的串联活性都显著高于通过羧基-胺结合。我们的结果表明，仿生定点固定化可以提供比化学特异性但非定向附着更重要的功能优势，这是在混合有机-无机器件上重现整个生物途径的兴趣日益增加的情况下的一个重要战略见解。
